Combination therapy

Abstract

The present disclosure relates to a therapeutic combination for use in treatment of a subject in need thereof, the combination comprising the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-f actor, the components being formulated for administration either as a single combined composition or as separate compositions. Also disclosed is a method of treatment making use of the disclosed combination, and a pharmaceutical product comprising the disclosed combination. The combination is particularly useful for the treatment of neurological damage caused by trauma or injury. A specific combination comprises glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), pyruvate (Pyr), oxaloacetate (OxAc), and pyridoxal phosphate (PLP).

Description

[0001] COMBINATION THERAPY

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to combination therapy of neurological conditions.

[0004] BACKGROUND

[0005] Neurotrauma leads to a rapid elevation of extracellular glutamate levels in the central nervous system (CNS). This elevation triggers excitotoxicity, which subsequently promotes inflammation, glial scar formation, and ultimately, neuronal death.

[0006] GENERAL DESCRIPTION

[0007] The present disclosure provides, in accordance with its first aspect, a method of treating a subject in need thereof, the method comprises administering to the subject a combination of components comprising (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined composition or as separate compositions.

[0008] The " subject in need" can be any subject that exhibits elevated glutamate levels in a bodily fluid as compared to a standard reference level, and / or is in predisposition of developing neurological damage that can be characterized by such elevated glutamate levels, and / or that has been subjected to neurological trauma.

[0009] The present disclosure provides, in accordance with a second of its aspects, a use of a combination of components for treatment of a subject in need thereof, the combination of components comprises (i) a first transaminase (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components beingformulated for administration either as a single combined composition or as separate compositions for simultaneous.

[0010] In accordance with yet its third aspect, the present disclosure provides a therapeutic combination for use in treatment of a subject in need thereof, the combination comprising the following components (i) a first transaminase (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined composition or as separate compositions.

[0011] In accordance with a fifth aspect, the present disclosure provides a pharmaceutical product comprising a combination of the following components:

[0012] (i) a first transaminase

[0013] (ii) a second transaminase,

[0014] (iii) a first substrate to the first transaminase

[0015] (iv) a second substrate to the second transaminase, and

[0016] (v) a transaminase co-factor,

[0017] wherein the components are formulated for administration either as a single combined composition or as separate compositions.

[0018] In accordance with the first, second, third, fourth and fifth aspects, the combination of components are formulated for essentially simultaneous administration, i.e. essentially together, as defined hereinbelow.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0021] Figure 1 is a graph showing time-course of glutamate levels in blood from naive mice, in vitro, following incubation with combined blood glutamate scavenging (cBGS) treatment rGOT1 (4.5μg) / rGPT1 (7.47ug) / OxAc (0.03M) / Pyr (0.03M) / PLP(0.008uM) (Combined) vs. the individual enzyme with co-substrate and co-factor PLP treatments (namely rGOT1+OxAc or GPT+Pyruvate). Results are mean ± STDEV of 5 repetitions of each point *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 - difference between combined treatment (cBGS, “Combined”) to rGPTl+Pyr+PLP and rGOTl+OxAc+PLP, significant difference rGOTl+OxAc+PLP treatment to rGPTl+Pyr+PLP ##p<0.01, ###p<0.001. One-way analysis of variance [ANOVA] followed by the Tukey HSD test, a set to 5%).

[0022] Figures 2A-2C are bar graphs showing CSF glutamate levels following spinal cord injury (SCI) in various treatment groups. Figure 2A: CSF Glutamate levels one-day post compression spinal cord injury, and after two injections of treatments, while the first one was administered one hour after the injury; Figure 2B: CSF Glutamate levels two days post compression spinal cord injury, first injection four hours after SCI;

[0023] Figure 2C: CSF Glutamate levels one-day post contusion spinal cord injury, injection one-hour after SCI. Sham: no SCI; Control / Con: treatment with vehicle; rGOT: rGOT1+OxAc+PLP; Combined: rGOT1 / OxAc + rGPT1 / pyr + PLP. Results are presented as mean ± SEM (minimum of n=3 animals per group, *p<0.05, **p<0.01, ***p<0.001).

[0024] Figure 3 is a bar graph showing axonal survival seven weeks following moderate / severe Spinal cord injury (SCI). The treatment involved 5 days of administration of combined BGS and showed a significant increase in axonal survival compared to control. Groups: Control n=11; 5 injections combined n=11; 1 injection combined n=8; 5 injections rGOT (rGOT1+OxAc+PLP) (n=12). Combined: rGOT1 / OxAc + rGPT1 / pyr + PLP. The first injection was administered four hours post injury. Results are presented as the mean ± SEM (*p<0.05,*** p<0.001).

[0025] Figures 4A-4C are bar graphs showing lesion size and neuronal rescue following moderate / severe compression SCI. Seven weeks post moderate / severe compression SCI, NeuN density around the lesion (Figure 4A) and synaptophysin density around positive NeuN neurons (Figure 4B) were significantly increased in 5-day combined BGS-treated mice compared to control. The lesion area was significantly lower in 5-day combined BGS-treated mice compared to controls. (Figure 4C). Control (Con) n=8; 5-day combined (GOT+GPT) n=5. Results are presented as the mean ± SEM (*p<0.05, **p<0.01,*** p<0.001).Figures 5A-5D are graphs showing improved outcomes after severe contusion SCI, following combined BGS treatment that started one hour after the injury, one week after severe spinal cord contusion. Quantitative analysis of GFAP density (Figure 5A) and Ibal (Figure 5B) around the lesioned area. Quantitative analysis of Thy 1 -axons crossing the lesion site (Figure 5C). BMS test demonstrated a significant improvement of hind limbs motor ability in combined BGS treatment compared to vehicle-control group (Figure 5D). Results are presented as mean+SEM. One-way ANOVA (***p<0.001), (****p<0.0001). Mann-5 Whitney test (*p<0.05). CON: Control; rGOT1+rGPT1: rGOT1 / OxAc + rGPT1 / pyr + PLP, combined treatment.

[0026] Figures 6A-6C are graphs showing Basso Mouse Scale (BMS) score and Grid walking test up to two weeks after compression SCI and BGS treatments.

[0027] Figure 6A: shows motor function analysis following Spinal Cord Injury (SPI) with the first injection administered 1-hour post-injury. The combined BGS, 5 days of treatment (Combined 5 inj), significantly improved hind limb motor ability compared to the 5 days of only rGOT1+OxAc+PLP (rGOT 5 inj) and control (CON, n=6 animals per group). Figure 6B: BMS test demonstrated a significant improvement of hind limbs motor ability in 5 days combined cBGS (“combined”) treatment compared to 5 days rGOT1+OxAc +PLP (“rGOT 5 inj”) treatment or 1-day combined treatment with a single injection administered 4 hours post-injury (“combined 1 inj”). Results are presented as mean+SEM; Mann- Whitney test (*p<0.05, ***p<0.001). Figure 6C: Grid walking test demonstrated a significant improvement of hind limbs motor ability in combined 5 days of treatment (“Combined 5 inj) compared to 5 days rGOTl+OxAc+PLP treatment (“rGOT 5 inj) or 1-day combined treatment (“Combined 1 inj”), which also demonstrated a significant improvement compared to vehiclecontrol group already 24 hours after the treatment. The treatment started four hours post-injury. Results are presented as mean ± SEM; Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001).

[0028] Figures 7A-7D are graphs showing gait motor deficits analysis (catwalk) two weeks after compression Spinal Cord Injury (SCI), with the first injection administered 4 hours post injury. Figure 7A: Normal step sequence represented by regularity index: the only 5-day combined treated mice had a significant improvement compared to control mice. Figure 7B-C: Swing speed and stride length: only the 5-day combinedtreated mice had a significant improvement compared to the control mice. Figure 7D: Run average speed was significantly higher in the 5-day combined treatment compared to control and rGOTl+OxAc+PLP treatment. Statistical analyses were done only between groups that received SCI; the Naive group was used only as a reference on how uninjured mice perform in the catwalk. Results are presented as the mean ± SEM (Control n=11; Combined 5 inj n=11; Combined 1 inj n=8; rGOT1 5 inj (rGOT1+OxAc + PLP) n=12, *p<0.05, **p<0.01).

[0029] Figures 8A-8B are graphs showing motor function analysis up to three weeks after compression Spinal Cord Injury (SCI), with the first injection administered 8 hours post injury. Figure 8A: BMS test demonstrated a significant improvement of hind limbs motor ability in 5 days of combined treatment compared to all different groups as specified. Result presented as mean j^SEM; Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001; n=8). Figure 8B: BMS test demonstrated a significant improvement of hind limbs motor ability in a single combined treatment injection compared to other single injection treatments and control groups as specified. Result presented as mean j^SEM; Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001; n=8).

[0030] Figures 9A-9B are immunostaining and bar graphs of GFAP marker three weeks after Spinal Cord Injury (SCI), with the first injection administered 8 hours postinjury. Figure 9A: GFAP density immunostaining in vehicle-control mice and combined BGS treatment 5-day injections, representative images of lesion site of axons represented by Thyl and GFAP. Scale bar 200 mm. Figure 9B: Quantitative analysis of GFAP density around the lesioned area. Results are presented as mean j^SEM. Oneway ANOVA (***p<0.001; n=8 mice in each group).

[0031] Figures 10A-10C are bar graphs showing the strong neuroprotective effects of 5 days of combined treatment (“BGS”) at three weeks after moderate / severe compression SCI, with the first injection administered 8 hours post-injury. Figure 10A:

[0032] Quantitative analysis of MBP density in the white matter tracks of axons at the lesion site of control and combined “BGS”-treated groups demonstrated a significant reduction of myelin basic protein breakdown at the injury site of combined 5-day- treated mice vs. the control (“CON”) untreated group. Results presented as mean+SEM. One-way ANOVA (**p<0.01; n=8 mice per group). Figure 10B:

[0033] Quantitative analysis of NeuN-positive cells at the lesion site. The data showsignificantly more NeuN-positive cells in combined “BGS”-treated mice compared to vehicle-control. Figure 10C: Quantitative analysis of synaptophysin immunostaining density around NeuN-positive cells showed that combined treatment significantly reduced synaptic degeneration around motor neurons at the lesion site. Results are presented as mean+SEM. One way ANOVA (*p<0.05, ***p<0.001; n=8 in each group).

[0034] Figures 11A-11B are qPCR analyses of pro-inflammatory markers 72 hours after TBI. Figure 11A: shows that IL-1β expression was significantly lower in the combined BGS-treated mice (“combined”) compared to the control group (CON).

[0035] Figure 11B: CX3CR1 expression was significantly lower in combined BGS-treated mice (“combined”) compared to the control group. Results are presented as the mean of fold change ± SEM. A minimum of n=4 animals per group and a minimum of three technical replicates; one-way ANOVA, **p<0.01, ****p<0.0001.

[0036] Figures 12A-12C are graphs showing motor and cognitive function analysis one week after a single moderate Traumatic Brain Injury (TBI). Figure 12A: presents Grip strength measurements in naive mice (NAIVE; n=11), vehicle-control (CON; n=5), and the combined “BGS” treated (BGS; n=8) groups. The result is presented as mean+SEM; way ANOVA (**p<0.01). Figure 12B: shows a 1cm beam walking test demonstrating significantly longer travel time in the control group compared to BGS-treated and naive mice (pre-injected) (CON, n=5; BGS, n=6; NAIVE mice, n=4). Result presented as mean+SEM; One way ANOVA (*p<0.05, **p<0.01). Figure 12C: presents an open field test demonstrating a significant improvement in the combined “BGS”-treated mice (n=9) in their time spent in the center compared to control (n=4) animals. Naive mice are pre-injected. Result presented as mean+SEM; One-way ANOVA.

[0037] Figures 13A-13B are graphs showing demyelination of axonal tracts one week after a single moderate Traumatic Brain Injury (TBI). Figure 13A: provides quantitative analysis of MBP density in the white matter tracks of axons at the lesion side (injured, Ips) and in the contralateral side (uninjured, Cnt) in the vehicle-control group and in the combined “BGS”-treated group. Figure 13B: provides quantitation of the percentage of Thyl-YFP positive cells (neuronal cells) at the lesioned area out ofthe number of cells in the parallel contralateral side. Results are presented as mean+SEM of control (n=5) and BGS (n=5) animals. One-way ANOVA (***p<0.001).

[0038] Figures 14A-14B are bar graphs showing GFAP and Iba-1 markers one week after a single moderate TBI. Figure 14A: provides quantitative analysis of GFAP density around the lesioned area one week after TBI examination of glial scaring by measurement of GFAP density immunostaining. Figure 14B: provides a quantitative analysis of Ibal density around the lesioned area one week after TBI examination of microglia activation by measurement of Ibal density immunostaining. Results are presented as mean + SEM of control (n=5) and BGS (n=5) animals. One-way ANOVA (**p<0.01); (***p<0.001).

[0039] Figures 15A-15C are scatter plots of Fractional Anisotropy (FA) values in different brain areas of treated groups following Repetitive Mild Traumatic Brain Injury in rats (rmTBI). Scatter plots presented with the median and inter-quartile range. The dots represent the number of brain areas in that brain region. Each dot is the average of the values for that brain area in sham (controls) without head injury (n=8), rats hit three times and treated with saline vehicle (“vehicle”) (n=8), and rats hit three times and treated with the combined BGS (“BGS”) (n=8). Figure 15A: Basal Ganglia area, Figure 15B: Cortex, and Figure 15C: Prefrontal cortex. Analysis for statistical significance was performed using a One-Way ANOVA. Differences between groups were assessed through separate t-tests. Ns=not significant; *<0.05; ** <0.01.

[0040] Figures 16A-16D are graphs showing IBA-1 signaling following repeated mild traumatic brain injury (rmTBI) in specific brain regions, accompanied by diagrams of respective brain areas (Figure 16B and 16D). Figure 16A: combined BGS treatment (“BGS”) decreased IBA1 density in the prefrontal cortex (Ctx) / midline on bregma (Figure 16B) Figure 16C: combined BGS treatment (“BGS”) decreased IB Al density in the hippocampus brain area (Figure 16D), one week after rmTBI (Bonferroni's Multiple Comparison Test; n=6 animals in each group). Control without head injury; rats hit three times and treated with saline vehicle (“vehicle”); and rats hit three times and treated with the combined BGS (“BGS”)

[0041] Figures 17A-17B are graphs showing GFAP signaling following repeated mild traumatic brain injury (rmTBI) in specific brain regions, accompanied by diagrams ofrespective brain areas. Figure 17A: One week after rmTBI injury, GFAP density in the hippocampus was significantly reduced in BGS-treated rats compared to the vehicle group, and Figure 17B: One week after rmTBI injury, GFAP density in the hypothalamus was significantly reduced in BGS-treated rats compared to the control group. (n=6 in each group; *p= 0.05 two-tailed t-test with 95% confidence.

[0042] Figure 18A–18D show representative MRI images and quantitative analyses of edema and lesion size following traumatic brain injury and treatment with single or combined BGS. Figure 18A: Representative T2-weighted coronal MRI images from each experimental group (Control, cBGS-treatment, rGOTl + OxAc + PLP, rGPTl + Pyr + PLP) acquired at 24 h, 48 h, and 1 week post-injury. Mice received the first injection of the assigned treatment 4 h post-injury. Images illustrate the temporal evolution of edema and lesion morphology across treatment groups. Figure 18B-18C:

[0043] Quantification of edema volume at 24 h (Figure 18B) and 48 h (Figure 18C) postinjury in male and female mice (n = 8 per group). Data are presented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test comparing cBGS-treatment, Control, rGOTl + OxAc + PLP, rGPTl + Pyr + PLP, and combined groups. ***P < 0.001; ****p < 0.0001; ****p < 0.0001; *P < 0.05; **P < 0.01; *P < 0.05. ns: not significant. Figure 18D: Quantification of lesion size at 1 week post-injury in male and female mice (n = 8 per group). Data are presented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test comparing cBGS-treatment, Control, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP groups. *P < 0.05; **P < 0.01. ns: not significant.

[0044] Figure 19A-19B are graphs showing beam-walking performance at 48 h, and anxiety-like behavior at 1 week following traumatic brain injury and BGS treatments.

[0045] Figure 19A: Graph showing the percentage of missteps on a 1-cm beam at 48 h postinjury in male and female mice (n = 8 per group). Mice received the first injection of the assigned treatment 4 h post-injury. Data are presented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test comparing cBGS-treatment, Control, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP groups. **** P < 0.0001; ***p < 0.001; *P < 0.05.

[0046] Figure 19B: Anxiety-related exploratory behavior assessed by the anxiety circle test at1 week post-injury in male and female mice (n = 8 per group). Data are presented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test comparing cBGS-treatment, Control, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP groups. **P < 0.01.

[0047] Figure 20A-20B are graphs showing neuronal survival and white matter integrity four weeks after traumatic brain injury and BGS treatment. Figure 20A:

[0048] Neuronal survival assessed by Thyl immunostaining four weeks after TBI in vehicle control, cBGS, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP treated mice. Mice received the first injection of the assigned treatment 4 h post-injury. Quantitative analysis of Thyl density was performed in the perilesional area. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using oneway ANOVA, with p values indicated in the figure. Figure 20B: Myelin basic protein (MBP) density in white matter axonal tracts at the lesion site compared with the contralateral hemisphere of the same mouse, four weeks after TBI. Quantitative analysis is presented as mean ± SEM (n = 6 animals per group). Statistical analysis was performed using one-way ANOVA (*P < 0.05).

[0049] Figure 21A–21C are bar graphs showing glial scarring and neuroinflammation four weeks after traumatic brain injury and BGS treatments. Figure 21A: quantitative analysis of astrogliosis assessed by GFAP immunostaining four weeks after TBI in vehicle-control, combined treatment, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP mice. GFAP density was quantified in the perilesional area. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure. Figure 21B: Microglial activation assessed by Ibal immunostaining four weeks after TBI. Ibal density was quantified in the perilesional area. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure. Figure 21C: Glial scar-associated extracellular matrix deposition assessed by CS56 immunostaining four weeks after TBI. CS56 density was quantified in the perilesional area. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure.Figure 22A-22B are graphs showing Beam-walking test performance at 24 and 48 h following traumatic brain injury and delayed BGS treatment. Figure 22A:

[0050] Percentage of missteps assessed by the beam-balance test (BBT) on a 1-cm beam at 24 h and 48 h post-injury in male and female mice (n = 8 per group). Mice received the first injection of the assigned treatment 8 h post-injury. Figure 22B: Percentage of missteps on a 1-cm beam at 48 h post-injury in male and female mice from an expanded cohort (total n = 38). Mice received the first injection of the assigned treatment 8 h postinjury. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons test comparing cBGS-treatment, Control, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP groups. *P < 0.05; **P < 0.01.

[0051] Figure 23A-23B are graphs showing neuronal survival and white matter integrity four weeks after traumatic brain injury and delayed BGS treatment. Mice received the first injection of the assigned treatment 8 h post-injury. Figure 23A:

[0052] Neuronal survival assessed by Thyl immunostaining four weeks after TBI in vehicle control, cBGS, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP treated mice. Mice received the first injection of the assigned treatment 8 h post-injury. Quantitation of the percentage of Thyl-YFP positive cells in the lesioned area out of the number of cells in the parallel contralateral side. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure. Figure 23B: Myelin basic protein (MBP) density in white matter axonal tracts at the lesion site compared with the contralateral hemisphere of the same mouse, four weeks after TBI. Quantitative analysis is presented as mean ± SEM (n = 6 animals per group). Statistical analysis was performed with p-values indicated in the figure.

[0053] Figure 24A-24B are bar graphs showing glial scarring and neuroinflammation four weeks after traumatic brain injury and delayed BGS treatments. Figure 24A: quantitative analysis of astrogliosis assessed by GFAP immunostaining four weeks after TBI in vehicle-control, combined treatment, rGOTl + OxAc + PLP, and rGPTl + Pyr + PLP mice. GFAP density was quantified in the perilesional area versus control untreated mice. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure.Figure 21B: Microglial activation assessed by Ibal immunostaining four weeks after TBI. Ibal density was quantified in the perilesional area. Data are presented as mean ± SEM (n = 5 animals per group). Statistical analysis was performed using one-way ANOVA, with p values indicated in the figure.

[0054] DETAILED DESCRIPTION

[0055] The present disclosure is based on the finding that treatment of an animal subjected to neurological damage with a combination of components comprising two different transaminase enzymes and two respective substrates, and a transaminase cofactor provided a therapeutic benefit that was greater than that observed following treatment with a single transaminase enzyme and its corresponding substrate.

[0056] It was further unexpectedly observed that the disclosed combination, namely, comprising two transaminase enzymes, two corresponding substrates, and a transaminase co-factor, was effective in treating neurological damage when administered to an animal model even after more than 4 hours post injury, e.g. at least 5 hours post injury, which is considered by those versed in the art a delayed time point for treatment following injury.

[0057] Additionally, as shown herein, unexpectedly, administration of the herein disclosed combination, namely, comprising two transaminase enzyme, the two respective substrates and the transaminase co-factor, at such delayed time points (i.e. more than 4 or even 5 hours post injury) resulted in at least one improved therapeutic parameter as compared to the same therapeutic parameter after administration of a treatment comprising only a single transaminase enzyme and its corresponding substrate (along with the co-factor) or as compared to a standard reference parameter or value. In the context of the present disclosure, when referring to a “standard reference”, it is to be understood to refer to a defined, measurable parameter or value that is established based on a recognized reference standard, control, or baseline, including a parameter or value obtained upon administration of the reference group (including no treatment, as a reference group), and against which other parameters or values are compared for the purpose of performance assessment, wherein such performance assessment is based on one or more quantitative or qualitative readouts,including levels of biological or biochemical markers, physiological or pathological characteristics, behavioral tests, functional assays, or combinations thereof.

[0058] Yet, a further unexpected finding was exhibited by the lack of observed side effects upon treatment with the disclosed combination of components.

[0059] Thus, a unique and surprising therapeutic combination is now being disclosed, the therapeutic composition comprising the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-f actor.

[0060] As disclosed herein, and in accordance with some preferred examples, the therapeutic combination is particularly advantageous for treatment of neurological damage, and the therapeutic combination can be administered or formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions for administration essentially together.

[0061] The present disclosure thus provides, in accordance with a first of its aspects, a method of treatment. The method involves administering to a subject in need of treatment, a combination comprising at least the following components: (i) a first transaminase (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions for essentially simultaneous (i.e. essentially together) administration. This first aspect is referred to, herein, at times, as the “ method aspect”.

[0062] The present disclosure provides, in accordance with a second of its aspects, a use of a therapeutic combination for treatment of a subject in need of treatment, the combination comprises the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase and (v) a transaminase co-factor, the components being formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions. This second aspect is referred to, herein, at times, as the “use aspect”.Further, the present disclosure provides, in accordance with a third of its aspects, a therapeutic combination for use in treatment of a subject in need of treatment, the combination comprising the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase and (v) a transaminase co-factor, the components being formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions for essentially simultaneous administration (i.e. essentially together, as defined herein). This third aspect is referred to, herein, at times, as the “ combination for use aspect”.

[0063] Yet further, the present disclosure provides, in accordance with a fourth aspect, a pharmaceutical product comprising a combination of components:

[0064] (i) a first transaminase

[0065] (ii) a second transaminase,

[0066] (iii) a first substrate to the first transaminase,

[0067] (iv) a second substrate to said second transaminase, and

[0068] (v) a transaminase co factor,

[0069] wherein the components are formulated for administration either as a single combined pharmaceutical composition or as two or more separate pharmaceutical compositions. This fourth aspect is referred to, herein, at times, as the “product aspect.

[0070] All disclosed aspects, namely, the method aspect, the use aspect, the combination for use aspect and the product aspect, require the combination per se of the listed components including the first transaminase, second transaminase, first substrate to the first transaminase, and second substrate to the second transaminase, and a co-factor to the transaminase. This combination is referred to herein as the “combination per se” or by the abbreviations of combined blood glutamate scavenging “cBGS”, or even the abbreviations “combined BGS”, “combined”, “BGS”, “GOT+GPT”.

[0071] The combination per se, according to the first, second, third and / or fourth aspect, comprises two different transaminases.In the context of the presently disclosed first, second, third and / or fourth aspect, the term “transaminase” has its commonly acceptable meaning, namely, an enzyme, also known by its name, aminotransferases, that facilitate the transfer of an amino group from an amino acid to a keto acid.

[0072] In the context of the presently disclosed first, second, third and / or fourth aspect, transaminase is one that relies on alanine and / or aspartate as the amino acid donor.

[0073] In accordance with the presently disclosed first, second, third and / or fourth aspect, the transaminase is one using glutamate as the amino group donor, namely, glutamate is the substrate in the transamination reaction it catalyzes.

[0074] In accordance with the presently disclosed first, second, third and / or fourth aspect, the transaminase in the combination per se is glutamate pyruvate transaminase (GPT).

[0075] In accordance with the presently disclosed first, second, third and / or fourth aspect, the GPT in the combination per se is any one of glutamate pyruvate transaminase 1 (GPT1) and glutamate pyruvate transaminase 2 (GPT2).

[0076] Genes encoding GPT, including GPT1 and GPT2 as well as amino acid sequences of GPT, including that of GPT1 and GPT2 are known in the art and can be found, inter alia, in National Center of Biotechnology Institute (NCBI) GeneBank.

[0077] In accordance with the presently disclosed first, second, third and / or fourth aspect, the GPT in the combination per se is GPT1.

[0078] In some examples of the presently disclosed first, second, third and / or fourth aspect, the accession numbers of the human GPT may be NM 005309.3 or NM OO 1382664.1 and encodes the amino acid sequence having the accession numbers NP_005300.1 or NP_001369593.

[0079] In some examples of the presently disclosed first, second, third and / or fourth aspect, the human GPT protein may comprise the amino acid sequence as denoted by SEQ ID NO: 1, or any variants, homologues or derivatives thereof. In some further embodiments, the human GPT protein may be encoded by a nucleic acid sequence comprising the nucleic acid sequence as denoted by SEQ ID NO:2 or SEQ ID NO:3, or any other variants, homologues or derivatives thereof.In accordance with the presently disclosed first, second, third and / or fourth aspect, the transaminase in the combination per se is glutamic-oxaloacetic transaminase (GOT).

[0080] In accordance with the presently disclosed first, second, third and / or fourth aspect, the GOT in the combination per se is any one of glutamic-oxaloacetic transaminase 1 (G0T1) or glutamic-oxaloacetic transaminase 2 (G0T2).

[0081] Genes encoding GOT, including G0T1 and GOT2 as well as amino acid sequences of GOT, including that of GOT1 and GOT2 are known in the art and can be found, inter alia, in National Center of Biotechnology Institute (NCBI) GeneBank.

[0082] In accordance with the presently disclosed first, second, third and / or fourth aspect, the GOT in the combination per se is GOT1.

[0083] In some examples of the presently disclosed first, second, third and / or fourth aspect the accession number of the human G0T1 is NM 002079.3 and encodes the amino acid sequence having the accession number NP_002070.1.

[0084] In some examples of the presently disclosed first, second, third and / or fourth aspect, the human G0T1 protein may comprise the amino acid sequence as denoted by SEQ ID NO: 4, or any variants, homologues or derivatives thereof. In some further embodiments, the human GOT1 protein may be encoded by a nucleic acid sequence comprising the nucleic acid sequence as denoted by SEQ ID NO: 5, or any variants, homologues or derivatives thereof.

[0085] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se comprises a combination of GOT (as described herein) and GPT (as described herein). It is to be appreciated that when referring herein to GOT and / or GPT it is to be understood to generally referring to any type of GOT and / or GPT as defined herein, unless otherwise stated.

[0086] It should be appreciated that the presently disclosed first, second, third and / or fourth aspect can also encompass variants or derivatives of the GPT or GOT polypeptides and any polypeptides that are substantially identical or homologue to the polypeptides encoded by the nucleic acid sequences described herein. The term "derivative" or "variant" is used herein to define amino acid sequences (e.g.polypeptides), with any insertions, deletions, substitutions and / or modifications to the native amino acid sequences of the transaminase that yet retains transaminase activity.

[0087] It should further be appreciated that the terms “ derivative" or "variant" can encompass orthologs and / or homologues of GPT or GOT from non-human species, including non-human mammalian species, that exhibit equivalent transaminase activity. Proteins orthologs or homologues having a sequence homology or identity to the transaminase of interest in accordance with the presently disclosed first, second, third and / or fourth aspect, specifically, the enzymes GPT1 or G0T1 and / or variants described herein, may share at least 70%, and specifically 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically as compared to the entire sequence of the original native proteins, for example, any of the proteins that comprise the amino acid sequence as denoted by any one of SEQ ID NO: 1 or SEQ ID NON. Specifically, homologs that comprise or consists of an amino acid sequence that is identical in at least 70%, at least 75% and specifically 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to the entire polypeptides that comprise the amino acid sequence as denoted by SEQ ID NO: 1 or SEQ ID NON and any derivatives, homologues and variants thereof.

[0088] In particular, the variant or derivative shares at least 90% amino acid sequence identity, preferably at least 95%, 96%, 97%, 98% or 99% identity, with the amino acid sequence of GPT1 or GOT1, as set forth in SEQ ID NO: 1 or SEQ ID NO: 4, and retains the transaminase activity of the respective native GPT1 or GOT1 transaminase. In some examples of the presently disclosed first, second, third and / or fourth aspect, derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions, deletions or substitutions of amino acid residues. It should be appreciated that by the terms "insertion / s", "deletion / s" or "substitution / s", as used herein it is meant any addition, deletion or replacement, respectively, of amino acid residues to the native polypeptides, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertion / s, deletion / s or substitution / s may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertion / s, deletion / s or substitution / s may occur in any position of the variant or derivative, as well as in any of the N' or C termini thereof.Thus, in some examples of the presently disclosed first, second, third and / or fourth aspect, the present disclosure makes use of the specifically referenced transaminase, as well as any variants or derivatives thereof, specifically a derivative that comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions to the amino acid sequences as denoted by any one of SEQ ID NO: 1 or SEQ ID NON.

[0089] The combination per se, forming part of the presently disclosed first, second, third and / or fourth aspect, also comprises at least two different substrates to transaminases, a first substrate of the first transaminase, and a second substrate of the second transaminase.

[0090] In the context of the presently disclosed first, second, third and / or fourth aspect, the term “ substrate to transaminase" has its commonly acceptable meaning, namely, a compound capable of participating in a transamination reaction catalyzed by a transaminase enzyme, wherein an amino group is transferred from an amino acid donor, such as glutamate, to an acceptor molecule, specifically an a-keto acid acceptor.

[0091] There are numerous known substrates for transaminases.

[0092] In some examples of the presently disclosed first, second, third and / or fourth aspect, the first and second transaminases in the combination per se are enzymes that act on glutamate, as the amino group donor, and the, respectively, first and second substrates in the combination per se, are compounds that serve as amino group acceptors, other than glutamate, i.e. are not glutamate.

[0093] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase GPT or GOT and the substrate for GPT or GOT is any compound that is capable of acting as an a-keto acid acceptor in the corresponding transamination reaction.

[0094] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GPT and the substrate is any compound capable of accepting an amino group from glutamate in a reaction catalyzed by GPT, to form the corresponding a-amino acid product.

[0095] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GPT and the substrate is one identified by its ability toundergo a transamination reaction catalyzed by GPT under physiological or in vitro conditions, forming alanine or a structurally analogous a-amino acid as the product.

[0096] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GPT and the substrate is selected from pyruvate, a-ketobutyrate, glyoxylate, or structurally similar a-keto acids or derivatives thereof that can act as amino group acceptors.

[0097] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GPT and the substrate is pyruvate.

[0098] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GOT and the substrate is any compound capable of accepting an amino group from glutamate in a reaction catalyzed by GOT to form the corresponding a-amino acid product.

[0099] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GOT and the substrate is one identified by its ability to undergo a transamination reaction catalyzed by GOT under physiological or in vitro conditions, forming aspartate or a structurally analogous a-amino acid as the product.

[0100] In some examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GOT and the substrate is selected from oxaloacetate, a-ketoglutarate, or structurally similar a-keto acids or derivatives thereof that can act as amino group acceptors.

[0101] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the transaminase is GOT and the substrate is oxaloacetate.

[0102] In some preferred examples of the presently disclosed first, second, third and / or fourth aspects, the first transaminase is glutamate pyruvate transaminase (GPT) and the second transaminase, being different from the first transaminase, is glutamate oxaloacetate transaminase (GOT).

[0103] In some examples of the presently disclosed first, second, third and / or fourth aspect, the first substrate to the first transaminase is selected in accordance with the substrate specificity of the first transaminase, such that when the first transaminase isGPT, the substrate is pyruvate (Pyr), or when the first transaminase is GOT - the substrate is oxaloacetate (OxAc).

[0104] In some examples of the presently disclosed first, second, third and / or fourth aspect, the second substrate, being different from the first substrate, is selected in accordance with the substrate specificity of the second transaminase, such that when the second transaminase is GPT the substrate is pyruvate (Pyr), or when the second transaminase is GOT the substrate is oxaloacetate (OxAc).

[0105] In some examples of the presently disclosed first, second, third and / or fourth aspect, the first substrate to the first transaminase is pyruvate (Pyr) or oxaloacetate (OxAc).

[0106] In some examples of the presently disclosed first, second, third and / or fourth aspect, the second substrate, being different from the first substrate, is pyruvate (Pyr) or oxaloacetate (OxAc).

[0107] In accordance with the presently disclosed first, second, third and / or fourth aspect, the combination per se also comprises a co-f actor.

[0108] In accordance with the presently disclosed first, second, third and / or fourth aspect, the co-factor in the combination per se comprises or is a compound capable of forming a Schiff base with the transaminase enzyme, stabilizing reaction intermediates, and / or facilitating transfer of the amino group during a transamination reaction.

[0109] In some examples of the presently disclosed first, second, third and / or fourth aspect, the co-factor in the combination per se comprises or is a phosphorylated vitamin B6 form, derivative or analog, including, without being limited thereto, pyridoxal 5'-phosphate (PLP), Pyridoxine phosphate (PNP) or Pyridoxamine phosphate (PMP) or a precursor of any of the above capable of being converted in vivo to PLP or PMP.

[0110] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the co-factor in the combination per se comprises or is PLP.

[0111] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se comprises GPT and GOT as the transaminases, Pyr and OxAc as the respective substrates to these transaminases, and PLP as the cofactor.The presently disclosed combination per se was found to provide a therapeutic advantage, when the at least two transaminases and the at least two corresponding substrates, in the presence of the co-factor, are administered essentially together.

[0112] In the context of the presently disclosed first, second, third and / or fourth aspect, when referring to an administration “essentially together" or "essentially simultaneously" it is to be understood the disclosed components of the combination per se, namely the at least two different transaminases and the corresponding at least two substrates, and the transaminase co-factor can be formulated in the same pharmaceutical composition (i.e. in the same dosage form) or formulated separately in two or more different pharmaceutical compositions, but are administered within a time window that is less than about 2 hours, preferably less than about 1 hour.

[0113] In some examples of the presently disclosed first, second, third and / or fourth aspect, the treatment with the combination per se comprises administrations of the first substrate separate from the respective first transaminase and / or the second substrate is administered separately from the corresponding second transaminase.

[0114] In some examples of the presently disclosed first, second, third and / or fourth aspect, the treatment with the combination per se comprises administration of the first transaminase in a one pharmaceutical composition with the first substrate and the second transaminase in one other pharmaceutical composition with the second substrate, and the co-factor can be included in either or both pharmaceutical compositions. The two pharmaceutical compositions are administered essentially together.

[0115] In some examples of the presently disclosed first, second, third and / or fourth aspect, the treatment with the combination per se comprises administration of the first and second transaminases in one pharmaceutical composition, and administration of the first and second substrates in one other pharmaceutical composition, and the co-factor can be included in either or both pharmaceutical compositions. The two pharmaceutical compositions are administered essentially together.

[0116] In some examples of the presently disclosed first, second, third and / or fourth aspect, the treatment with the combination per se comprises a single pharmaceutical composition comprising the first transaminase, the first substrate to the firsttransaminase, the second transaminase and the second substrate of the second transaminase, and the co-factor.

[0117] In some examples of the presently disclosed first, second, third and / or fourth aspect, the treatment with the combination per se comprises a single pharmaceutical composition comprising GPT and GOT as the transaminases, Pyr and OxAc as the respective substrates to these transaminases, and PLP as the co-factor.

[0118] In some examples of the presently disclosed first, second, third and / or fourth aspect, the components of the combination per se are formulated in amounts that are effective to provide a desired therapeutic effect.

[0119] In the context of the presently disclosed first, second, third and / or fourth aspects, the term “desired therapeutic effect' refers to a beneficial therapeutic outcome as determined by a physician or other qualified healthcare professional, based on medically acceptable and clinically relevant parameters. Without limitation, a desired therapeutic effect may include any one or more of the following: reduction of extracellular glutamate levels; reduction of excitotoxic neuronal death or apoptosis; preservation or improvement of neuronal viability, synaptic integrity, or axonal integrity; reduction of tissue damage; reduction of lesion size; attenuation of neuroinflammatory responses, including modulation or reduction of microglial activation; reduction of astrocytic gliosis; reduction of scarring; stabilization or restoration of blood-brain barrier integrity; reduction of cerebral edema or intracranial pressure; reduction of oxidative stress or reactive oxygen species levels; improvement in neurotransmitter homeostasis; promotion of neuroprotection, neurogenesis, synaptic plasticity, or axonal regeneration; improvement in one or more neurological, cognitive, and / or motor functions; reduction in seizure frequency or severity, where applicable; delay in the progression of neurological damage; amelioration of one or more symptoms associated with a neurological condition or injury; improvement in electrophysiological parameters; improvement in imaging-based biomarkers indicative of tissue integrity or inflammation; improvement in functional recovery; and / or improvement in quality of life and activity of daily living (ADL).

[0120] In some preferred examples of the presently disclosed first, second, third and / or fourth aspects, the desired therapeutic effect, assessed using conventional and clinicallyestablished parameters, includes any one or combination of reduction of extracellular glutamate levels; reduction of tissue damage and / or lesion size; attenuation of neuroinflammatory responses; reduction of astrocytic gliosis and / or scarring; improvement in one or more neurological, cognitive, and / or motor functions; delay in the progression of neurological damage; amelioration of one or more symptoms associated with the neurological condition or injury; improvement in functional recovery; and / or improvement in quality of life.

[0121] As noted above, the combination per se is administered in an amount that is effective to provide the desired therapeutic effect.

[0122] As used herein, an “amount effective” refers to an amount of the combination per se, or of the amounts of the components thereof, that is sufficient to provide a measurable therapeutic difference in the evaluated relevant parameter (e.g. one or more of the parameters listed above).

[0123] In some examples of the presently disclosed first, second, third and / or fourth aspect, the amount would be considered effective if it is sufficient to cause one or more of the following: reduce glutamate levels in blood and / or cerebrospinal fluid, reduce lesion size, reduce tissue damage, reduce neuroinflammatory responses, reduce astrocytic gliosis or scarring, improve neurological, cognitive, and / or motor function.

[0124] The amount may vary depending on one or more factors including, without limitation, the nature and / or severity of the condition to be treated (preferably neurological condition to be treated); the nature and / or severity of an injury; the timing of administration relative to the occurrence of the injury; the age, sex, weight, and overall medical condition of the subject; the route of administration; and the specific pharmaceutical composition, including the combination per se and / or its individual components.

[0125] In some examples of the first, second, third and / or fourth aspect of the present disclosure, the amount is also an amount in which any potential adverse or undesirable effects (if any of such exist) are outweighed by the therapeutically beneficial effects achieved.

[0126] An effective amount may be administered as a single administration or as multiple administrations over time. An effective amount of one or more components ofthe combination may be an amount that, when administered together with one or more additional components of the combination per se, results in the desired therapeutic effect through the combined action of the components. Accordingly, a particular component may be administered at an amount that is effective in the context of combination therapy, even if that amount would not produce the desired therapeutic effect when the component is administered alone.

[0127] In some examples of the presently disclosed first, second, third and / or fourth aspect, the amounts of the first transaminase and the first substrate, and the amounts of the second transaminase and the second substrate, in the combination per se are sufficient to provide a therapeutic effect that is greater or higher (in accordance with medically acceptable considerations) than the effect provided by administration of either enzyme-substrate pair alone.

[0128] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se provides an enhanced therapeutic effect relative to treatment with transaminase and its corresponding substrate, even when the single transaminase - substrate is administered at equal or higher amounts / concentrations.

[0129] In some examples of the presently disclosed first, second, third and / or fourth aspect, the relative amounts of each transaminase and its corresponding substrate in the combination per se are selected in accordance with ratios compatible with the desired transaminase-catalyzed reactions.

[0130] It has been shown that the combination per se provides a therapeutic effect (according to acceptable considerations) greater than the effect achieved with treatment with GOT + OxAc + PLP or GPT + Pyr + PLP.

[0131] In some examples of the presently disclosed first, second, third and / or fourth aspect, an enhanced therapeutic effect of the combination per se is observed under conditions in which the amount of at least one of the individual component (i.e. transaminase and / or substrate) in the combination per se is lower than the amount required to provide the same therapeutic effect when the component is administered as part of a single enzyme and substrate treatment.

[0132] As used herein, comparative terms such as “greater”, “improved”, “enhanced”, “lower”, “reduced”, and the like refer to a measurable difference in at least onetherapeutic parameter relative to a reference treatment, as determined using experimental or clinical assessment methods known in the art. In some examples, such terms correspond to a measurable improvement or reduction relative to a single enzyme-substrate treatment. In some examples, the measurable improvement or reduction may be considered to be associated with a synergistic relationship between the components.

[0133] In the context of the present disclosure, the term “enhanced / improved / greater / lower / reduced” refers to a therapeutic effect obtained upon administration of the disclosed combination of components that exceeds the effect expected from a purely additive contribution of the individual components when administered alone, as determined by comparison to the sum or aggregate of the effects achieved by each individual component or individual enzyme-substrate pair under otherwise comparable conditions. An enhanced therapeutic effect, as used herein, does not require a finding of pharmacological synergism, but rather indicates that the observed effect of the combination cannot be fully accounted for by a simple arithmetic addition of the individual effects and reflects a non-additive improvement in at least one measured therapeutic parameter.

[0134] In some examples of the presently disclosed first, second, third, and / or fourth aspects, the therapeutic effect obtained with the combination per se provides a statistical difference to the reference treatment. In this connection, the term "statistical difference" refers to a difference determined using conventional statistical tests commonly employed in the art. These tests may include, but are not limited to, parametric tests such as t-tests (e.g., paired or unpaired t-tests), analysis of variance (ANOVA), and regression analyses, as well as non-parametric tests like the Mann-Whitney U test, Kruskal-Wallis test, and Wilcoxon signed-rank test. The choice of the statistical test is determined based on the nature of the data (e.g., distribution, sample size, and homogeneity of variance) and the specific experimental design. Statistical significance is typically assessed at a predetermined significance level (e.g., p < 0.05), as is standard in the field.

[0135] In some examples of the presently disclosed first, second, third, and / or fourth aspect, the combination per se provides a therapeutic effect that is statistically greaterthan the effect achieved when treatment is with a glutamate-scavenging agent comprising GOT+OxAc+PLP or GOT + OxAc + PLP.

[0136] For example, to achieve a same therapeutic effect, if the reference treatment comprises GOT+OxAc+PLP, and the combination per se comprises GOT+ OxAc+GPT+Pyr+PLP, the concentration of GOT and OxAc in the reference treatment would be typically greater, as defined herein, or equal to the combined concentration of GOT and GPT, and the combined concentration of OxAc and Pyr, respectively, in the combination per se.

[0137] The combination per se, forming part of the first, second, third and fourth aspects of the presently disclosed subject matter is used for providing a therapeutic effect in a subject, e.g. treatment of a subject in need of achieving a desired therapeutic effect, as described herein.

[0138] In the context of the presently disclosed first, second, third and / or fourth aspect, providing a therapeutic effect, collectively referred to by the terms “treating, “treatment” and the like is used herein to refer to obtaining a desired pharmacological and / or physiological effect in a subject. Such treatment may relate to an existing condition or neurological damage, as well as prophylactic in nature, intended to prevent, delay, or reduce the severity of such condition or damage, as further detailed hereinbelow.

[0139] A subject is considered successfully “treated” if one or more of the foregoing therapeutic effects is achieved, whether partially or completely, and whether directly or indirectly through the combined action of the components of the combination per se as disclosed herein.

[0140] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is for treatment of neurological condition involved with elevated glutamate levels (extracellular levels).

[0141] As used herein, in the context of the presently disclosed first, second, third and / or fourth aspect, the term “ elevated glutamate levels” refers to glutamate levels that are increased relative to acceptable reference physiological baseline levels. Without being limited thereto, glutamate levels would be determined from any one of bloodlevel, plasma level, serum level, cerebrospinal fluid (CSF) level, brain tissue level, or level in the interstitial fluid of the central nervous system.

[0142] As used herein, a “neurological condition" refers to a disorder, injury, or disease affecting the central or peripheral nervous system.

[0143] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is for treatment of neurological condition caused by trauma. In some examples of the presently disclosed first, second, third and / or fourth aspect, when the neurological condition is caused by trauma, it exhibits elevated glutamate levels.

[0144] In some examples of the presently disclosed first, second, third and / or fourth aspect, when the neurological condition caused by trauma, it encompasses “neurological damage". As used herein, a “neurological damage" refers to structural and / or functional impairment of the central and / or peripheral nervous system.

[0145] In the context of the presently disclosed first, second, third and / or fourth aspect, when referring to treatment of a neurological damage caused by trauma, the term "treating", "treatment", and the like are used herein to refer to obtaining a desired pharmacological and / or physiological effect with respect to a neurological damage caused or that may be caused by trauma. Such treatment may be prophylactic, therapeutic or both, and may include preventing, delaying, reducing, or partially or fully reversing neurological damage or one or more symptoms associated therewith.

[0146] In the context of the presently disclosed invention, the term "trauma" refers to a physical chemical, or insult to the nervous system that results in neurological damage. Trauma includes, without being limited thereto, blunt force impact, blast -related injury, penetrating injuries, acceleration-deceleration injury, ischemic or hemorrhagic events such as stroke, exposure to neurotoxic substances, or conditions resulting from surgical or therapeutic interventions.

[0147] Further, the term "trauma" covers acute, sub-acute, or chronic forms of trauma, with potential effects ranging from transient dysfunction to permanent structural and / or functional impairment of the central and / or peripheral nervous system.

[0148] The neurological damage being treated according to the presently disclosed first, second, third and / or fourth aspect, can be of any level of severity as known in theart. For example, the damage can be mild neurological damage, moderate neurological damage, severe neurological damage or progressive neurological damage, as determined using clinical, functional, imaging, and / or biochemical assessment methods known in the art.

[0149] For example, the severity of neurological damage caused by trauma can be assessed using parameters and protocols well-known in the art, including clinical scales such as the Glasgow Coma Scale (GCS) for evaluating consciousness, the National Institutes of Health Stroke Scale (NIHSS) for neurological impairment, American Spinal Injury Association American Impairment Scale (ASIA) for spinal cord injury and the Extended Glasgow Outcome Scale (GOSE) for outcomes. Imaging modalities like CT or MRI, including advanced techniques such as Diffusion Tensor Imaging (DTI), assess structural damage, while functional evaluations such as neuropsychological tests and the Functional Independence Measure (FIM) evaluate cognitive and motor deficits. Biomarkers like S100B, Iba1, GFAP, CS56 and / or MBP may also be used to gauge injury severity. These standardized methods provide objective criteria to define the extent of damage, ranging from mild, moderate, and severe to progressive, based on widely accepted clinical and research protocols.

[0150] In some more specific examples of the presently disclosed first, second, third and / or fourth aspect, the neurological damage caused by trauma is selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, hemorrhagic brain injury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition.

[0151] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the neurological damage is SCI.

[0152] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the neurological damage is TBI.

[0153] In the context of the presently disclosed subject matter, the abbreviations “SCI” and “TBI”, as used herein, include injuries of varying severity and etiology, including mild, moderate, severe, acute, sub-acute, and chronic forms.In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the neurological damage is concussion. As used herein, “concussion” includes single or repeated concussive injuries, including, without being limited thereto, those occurring in athletic or sports-related settings.

[0154] In some preferred examples of the presently disclosed first, second, third and / or fourth aspect, the neurological damage is blast related neurological injury, or in short "blast injury".

[0155] As used herein, the term “blast injury" refers to injury to the nervous system resulting from exposure to a blast or explosive force, including injury caused by the primary blast wave (overpressure), as well as secondary or tertiary blast effects.

[0156] Blast injury typically affects the central and / or peripheral nervous system and may include, without limitation, blast-induced traumatic brain injury, spinal cord injury, injury to peripheral nerves, diffuse axonal injury, neurovascular damage, and associated neuroinflammatory responses.

[0157] Blast injury can occur with or without direct physical impact to the head or body and can result in acute, sub-acute, or chronic neurological damage.

[0158] In accordance with the presently disclosed first, second, third and / or fourth aspect, the combination per se can be administered using different administration regimens. Irrespective of the selected regimen, the regimen includes at least a first administration of the combination per se at a first time point within a defined treatment window.

[0159] A. Early (acute) first-administration regimen

[0160] In some examples of the presently disclosed first, second, third and / or fourth aspect, the administration regimen comprises a first administration of the combination per se at a first time point, the first time point being within a first treatment window of up to 4 hours post trauma.

[0161] In some examples of the presently disclosed first, second, third and / or fourth aspect, the early, first administration regime comprises at least one additional administration of the combination after the first treatment window. It is to be appreciated that the combination per se, in the at least one additional administrationmay be the same as or different from the combination administered in the first administration.

[0162] In some examples of the presently disclosed first, second, third and / or fourth aspect, the early first administration regimen comprises two or more additional administrations of the combination at respective two or more additional (different) time points after the first treatment window. It is to be appreciated that the combination per se, the two or more additional administrations can be the same as or different from the combination of the first administration, as well as different one from the other.

[0163] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the early first administration regime comprises two or more additional administrations, at least one of the two or more additional administrations is administered within a second treatment window that is from at least 6 hours; at times, at least about 8 hours; at times, at least about 10 hours; at times, at least about 12 hours, after the first treatment window and up to 48 hours post trauma.

[0164] In some examples of the presently disclosed first, second, third and / or fourth aspect, when the early first administration regime comprises at least one additional administration, the first of the at least one additional administration is within the second time window and with a time interval from the first treatment / administration of at least 10 hours, at times, of at least 15 hours; at times, of at least 20 hours.

[0165] In the context of the presently disclosed first, second, third and / or fourth aspect, when referring to a “ second time window" it is to be understood to encompass any time point starting 12 hours; at times, 13 hours; at times 14 hours post trauma (i.e. from the time the traumatic event occurred), and up to 48 hours post trauma.

[0166] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the early first administration regime comprises two or more additional administrations, in addition to the first administration, each additional administration is separate from any other additional administration by a time interval of at least 10 hours, at times, of at least 15 hours; at times, of at least 20 hours.

[0167] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the early first administration regime comprises two or more additional administrations, each additional administration is separate from any other additionaladministration by a time interval of between 10 hours and 48 hours; at times, by a time interval of between 20 and 48 hours; at times, by a time interval of between 25 and 48 hours; at times, by a time interval of between 30 and 48 hours.

[0168] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the early first administration regime comprises two or more additional administrations, the additional administrations are provided at time intervals of about 24 hours.

[0169] In some examples of the presently disclosed first, second, third and / or fourth aspect, the early first administration regime comprises daily administrations of the two or more additional administrations.

[0170] In some examples of the presently disclosed first, second, third and / or fourth aspect, the early first administration regime comprises more than two additional administrations, with at least one of the additional administrations being administered after the second time window. Thus, for example, the early first administration regime may comprise a first administration within 4 hours post trauma (i.e. within the first early administration window), a second administration about 24 hours post trauma, followed by daily administration of at least two more additional administrations (thus, in overall, there would be at least 4 administrations, preferably even at least 5 administrations).

[0171] In some examples of the presently disclosed first, second, third and / or fourth aspect, the early first administration regime comprises four additional administrations. Thus, for example, the early first administration regime may comprise a first administration within 4 hours post trauma, a second administration about 24 hours after the first administration, followed by daily administration of three or at least three additional administrations.

[0172] As noted above, each administration can include the same or a different compositions as defined herein.

[0173] B. Late first administration regime

[0174] It has now been found that the presently disclosed combination per se has an advantage) when the first administration of the combination per se is after the first treatment window (of up to 4 hours post trauma).Surprisingly, it has been found that the presently disclosed combination per se provides a therapeutic advantage even when the first administration is performed within a belated ("late") treatment window.

[0175] The ability to provide a therapeutic benefit when the first administration occurs within a late treatment window is of clinical importance, as patients with neurological damage caused by trauma frequently experience delays in recognition, diagnosis, triage, transport, or access to definitive medical care, such that treatment is often initiated several hours after the traumatic event.

[0176] It has been further found that providing the first administration of the combination disclosed herein within the late treatment window provides, at times, an improved effect when compared to treatment with a single pair of enzyme and substrate of a glutamate scavenging agent (e.g. the pairs being GOT + OxAc + PLP or GPT + Pyr + PLP).

[0177] Thus, in accordance with the presently disclosed first, second, third and / or fourth aspect, the combination per se is first administration of the combination per se is within a treatment window that is greater than 4 hours post trauma, namely, a "late time window".

[0178] In the context of the presently disclosed first, second, third and / or fourth aspects, the term "late treatment window" is to be understood to refer to a first administration of the combination per se taking place after more than 4 hours from the time of injury or traumatic event occurred and is thus considered to be later than the first time window according to the early first administration regime defined herein.

[0179] Thus, in some examples of the presently disclosed first, second, third and / or fourth aspect, the late - first administration regime comprises a first administration of the combination at a time point within a late treatment window that is greater than 4 hours post injury or trauma.

[0180] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is more than 4 hours and up to 36 hours post injury or trauma.In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is more than 6 hours and up to 24 hours post injury or trauma.

[0181] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is between 6 and 12 hours post injury or trauma.

[0182] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is between 6 and 10 hours post injury or trauma.

[0183] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is between 7 and 12 hours post injury or trauma.

[0184] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is between 8 and 12 hours post injury or trauma.

[0185] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late- first administration regime comprises a late first administration window that is between 8 and 10 hours post injury or trauma.

[0186] In some examples of the presently disclosed first, second, third and / or fourth aspect, the first administration according to the late- first administration regime is about 8 hours post injury or trauma.

[0187] When the late- first administration regime comprises a first administration within the late treatment window, it is preferable to provide at least one additional administration of the combination subsequent to the first administration.

[0188] In some examples of the presently disclosed first, second, third and / or fourth aspect, the at least one additional administration according to the late- first administration regime is formulated the same or different from the combination of the first administration according to the late-first administration regime.

[0189] In some examples of the presently disclosed first, second, third and / or fourth aspect, when the late-first administration regime comprises two or more additionaladministrations, the first of the additional administrations is administered within the second time window as herein defined.

[0190] The second time window in the context of the late-first administration regime has the same meaning as the second time window according to the early-first administration regime. Thus, in the context of the presently disclosed first, second, third and / or fourth aspect, when referring to a “second time window" according to the late-first administration regime, it is to be understood to encompass any time point starting 12 hours; at times, 13 hours; at times 14 hours post trauma (i.e. from the time the traumatic event occurred), and up to 48 hours post trauma.

[0191] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the late-first administration regime comprises a first administration at a late treatment window, and two or more additional administrations in addition to the first administration, additional administration is separate from any other additional administration by a time interval of at least 6 hours, at times, of at least 10 hours; at times, of at least 15 hours; at times, of at least 20 hours.

[0192] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the late first administration regime comprises two or more additional administrations, each additional administration is separate from any other additional administration by a time interval of between 6 hours and 48 hours; at times, with a time interval of between 8 hours and 24 hours; at times, with a time interval of between 10 hours and 48 hours; at times, with a time interval of between 20 and 48 hours; at times, with a time interval of between 25 and 48 hours; at times, with a time interval of between 30 and 48 hours.

[0193] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late first administration regime comprises two or more additional administrations, the additional administrations are provided at time intervals therebetween of about 24 hours.

[0194] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late-first administration regime comprises daily administrations of the additional administrations.In some examples of the presently disclosed first, second, third and / or fourth aspect, the late first administration regime comprises more than two additional administrations, with at least one of the additional administrations being administered after the second time window. Thus, for example, the late first administration regime may comprise a first administration about 8 hours post trauma or injury, a second administration (i.e. the first additional administration) about 24 hours after the first administration, followed by daily administration of at least two more additional administrations (thus, in overall, there would be at least 4 administrations, preferably even at least 5 administrations).

[0195] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late first administration regime comprises four additional administrations. Thus, for example, the treatment may comprise a first administration at least about 8 hours post trauma or injury, a second administration after about 24 hours from the first administration, followed by daily administration of three additional administrations.

[0196] In some examples of the presently disclosed first, second, third and / or fourth aspect, the late first administration regime comprises four additional administrations. Thus, for example, the treatment may comprise a first administration at least 4 hours post trauma or injury, a second administration after about 24 hours from the first administration, followed by daily administration of three additional administrations.

[0197] As shown in the non-limiting examples provided herein, the combination per se provided according to the late first administration regime exhibited an advantage over treatment with a single enzyme- substrate pair of glutamate scavenging agents, comprising a transaminase, a substrate to the transaminase and a transaminase cofactor.

[0198] Thus, in accordance with a preferred example, the presently disclosed first, second, third and / or fourth aspects comprises a first administration of the combination per se, preferably, the combination comprising GOT + OxAc + PLP or GPT + Pyr + PLP) at least 6 hours post trauma, preferably at least 7 hours post trauma, at least 8 hours post trauma, at least 12 hours post trauma, but not more than 36 hours post trauma) followed by daily administration of the combination of GOT + OxAc + PLP or GPT + Pyr + PLP for an additional 4 times.Irrespective of the treatment regime (be it the early or late first administration regime) the therapeutic effect can be determined by a change in one or more parameters, acceptable in the art.

[0199] In some examples of the presently disclosed first, second, third and / or fourth aspect, the therapeutic effect can be determined by a change in one or more parameters selected from the group consisting of: reduction of glutamate level in blood and / or cerebrospinal fluid (CSF), reduction in edema size and / or lesion size, reduction of at least one neuroinflammatory marker, reduction of astrocytic gliosis, reduction of scarring, improvement in cognitive and / or motor behavior, the change being determined relative to the same parameter prior to treatment with the combination per se.

[0200] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is preferably administered by injection or infusion.

[0201] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is administered by any one of intravenous administration, intramuscular administration or subcutaneous administration.

[0202] In some examples of the presently disclosed first, second, third and / or fourth aspect, where the treatment comprises more than one administration of the combination per se, the first administration (be it according to the early or late administration regime) is by intravenous administration and any additional administration is independently selected from the group consisting of intravenous, intramuscular and subcutaneous administration.

[0203] In some examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is formulated into a pharmaceutical composition suitable for oral administration.

[0204] In some examples of the presently disclosed product, the components of the product are formulated for administration as separate compositions, the first transaminase being in a same composition with its substrate (i.e. the first substrate) and the second transaminase being in a same composition with its substrate (i.e. the second substrate). The transaminase co-factor can be formulated into one or both of the separate compositions.In some examples of the presently disclosed product, the components of the product are formulated for administration as a single combined pharmaceutical composition comprising the first transaminase, the (first) substrate to the first transaminase, the second transaminase and the (second) substrate of the second transaminase and the transaminase co-factor.

[0205] The presently disclosed product typically also includes instructions for use of the combination per se. As exemplified herein, the instructions involve combined administration of two sets of a transaminase and respective substrates, and these instructions are shown to provide an unexpected advantage over administration of a single pair of transaminases and respective substrates (along with the co-factor).

[0206] The instructions associated with the disclosed product may include guidance on one or more of the following: frequency of administration, dosage of each component, duration of treatment with the combination, route of administration, and any preparatory steps required from the subject prior to treatment with the combination.

[0207] In some examples of the presently disclosed fourth aspect, the instructions, forming part of the disclosed product, allow for the use of the components of the product for treating of a neurological condition. In other words, the instructions provide the steps for treatment of a neurological condition, the neurological condition being as detailed and / or defined hereinabove.

[0208] In some examples of the presently disclosed product, the instructions are for treating a neurological condition caused by trauma.

[0209] In some examples of the presently disclosed product, the instructions specify administration of the combination per se at a first time point within a first treatment window extending up to 4 hours from the time of trauma or injury, i.e. according to the early first administration regime.

[0210] In some examples of the presently disclosed product, the instructions specify administration of the combination per se at a first time point within a first treatment window extending more than 4 hours from the time of trauma or injury, i.e. according to the late first administration regime.

[0211] In some examples of the presently disclosed product, the instructions specify administration of the combination per se at the first time point and at least oneadditional administration of the combination per se after the first treatment window (be it according to the early or late treatment regime), wherein the combination per se, administered in the additional administrations may be formulated the same or different from the combination administered in the first administration.

[0212] In some examples of the presently disclosed product, the instructions specify administration of the combination per se to include a first administration within the first treatment window (be it according to the early or late treatment regime) and two or more additional administrations of the combination per se at respective time points after the first treatment window, with at least one of the additional administrations occurring within a second treatment window, as defined hereinabove with respect to all disclosed aspects.

[0213] In some examples of the presently disclosed product, the instructions specify daily administration of the two or more additional administrations of the combination per se.

[0214] In some examples of the presently disclosed product, the instructions specify the route of delivery, as described hereinabove with respect to all presently disclosed aspects.

[0215] In some other examples of the presently disclosed first, second, third and / or fourth aspect, the combination per se is for treatment of a neurological condition that is not necessarily caused by trauma or injury. Such conditions that are not necessarily caused by trauma can be selected from the group consisting of migraine, epilepsy amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and Huntington’s disease.

[0216] LIST OF PARAGRAPHS

[0217] The following statements / paragraphs disclose features of the present disclosure. It is to be appreciated that any combination of these two or more of these paragraphs, or parts of paragraphs, constitute part of the invention and that there should be no limitation to the number of paragraphs that can be combined, as part of the presently disclosed subject matter:1. A method of treatment of a subject in need thereof, the method comprises administering to the subject a combination comprising the following components (i) a first transaminase (ii) a second transaminase that is different from the first transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, the second substrate being different from the first substrate, and (v) a transaminase co-factor, the combination being formulated for administration either as a single combined pharmaceutical composition or as separate compositions for essentially simultaneous administration.

[0218] 2. The method of paragraph 1, wherein the first transaminase and the second transaminase are selected from the group consisting of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

[0219] 3. The method of paragraph 1 or 2, wherein the first substrate and the second substrate are selected from pyruvate (Pyr) and oxaloacetate (OxAc).

[0220] 4. The method of any one of the preceding paragraphs, wherein the co-factor is pyridoxal phosphate (PLP).

[0221] 5. The method of any one of the preceding paragraphs, comprising administration of a combination comprising GPT as the first transaminase, GOT as the second transaminase, Pyr as the first substrate to the first transaminase, OxAc as the second substrate to the second transaminase, and PLP as the transaminase co-factor.

[0222] 6. The method of any one of the preceding paragraphs, wherein the treatment comprises administrations of the first substrate separately from the administration of the first transaminase and / or the second substrate separately from the second transaminase.

[0223] 7. The method any one of the preceding paragraphs, wherein the treatment comprises administration of the first transaminase in a same pharmaceutical composition with the first substrate and the second transaminase in a same pharmaceutical composition with the second substrate of the second transaminase. 8. The method of any one of the preceding paragraphs, wherein the treatment comprises administration of a single pharmaceutical composition comprising the first transaminase, the first substrate to the first transaminase, the second transaminase and the second substrate of the second transaminase, and the transaminase co-factor.9. The method of any one of the preceding paragraphs, wherein the treatment is for a neurological condition, preferably a neurological damage caused by trauma or injury.

[0224] 10. The method of at least the preceding paragraphs, wherein the treatment comprises a first administration of the combination at a first time point after the event of trauma, the first time point being within a first (early) treatment window comprising any time point from the traumatic event and up to 4 hours post trauma or injury.

[0225] 11. The method of any one of the preceding paragraphs, comprising a first administration of the combination at the first time point and at least one additional administration of the combination after the first treatment window, the combination of the at least one additional administration being formulated the same or differently from the combination of the first administration.

[0226] 12. The method of any one of the preceding paragraphs, comprising two or more of the at least one additional administration of the combination, at respectively two or more additional (different) time points after the first treatment window.

[0227] 13. The method of any one of the preceding paragraphs, wherein at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma or injury.

[0228] 14. The method of any one of paragraphs 1 to 9, comprising a first administration of the combination at a time point within a late treatment window that is greater than 4 hours post injury or trauma (i.e. the first treatment window is later than 4 hours). 15. The method of paragraph 14, wherein the late treatment window is up to 36 hours post injury or trauma.

[0229] 16. The method of paragraph 14 or 15, wherein the first administration is 6-12 hours post injury or trauma.

[0230] 17. The method of any one of paragraphs 14 to 16, comprising at least one additional administration of the combination subsequent to the first (late) administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first late administration.18. The method of any one of the preceding paragraphs, wherein, when having two or more of said at least one additional administration, the additional administrations are daily administrations of the combination.

[0231] 19. The method of any one of the preceding paragraphs, wherein the treatment provides an effect that is greater, as defined herein, than the effect achieved with treatment comprising a single transaminase, a single substrate to the transaminase and a transaminase co-factor.

[0232] 20. The method of any one of the preceding paragraphs, wherein the treatment with the disclosed combination provides an effect that is greater, as defined herein, than the effect achieved when the treatment is with GOT, OxAc, and PLP.

[0233] 21. The method of any one of the preceding paragraphs, providing a therapeutically and / or medically significant change in any one of the following parameters: reduction of glutamate level in blood and / or cerebrospinal fluid (CSF), reduction in edema size and / or lesion size, reduction of at least one inflammatory marker, reduction of astrocytic gliosis, reduction of scarring, improvement in cognitive and / or motor behavior, the change being determined in comparison to the parameter before treatment or in comparison with an acceptable reference (e.g. a medically acceptable reference). 22. The method of any one of the preceding paragraphs, wherein the treatment is of a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, haemorrhagic brain injury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition. 23. The method of any one of the preceding paragraphs, for treatment of SCI. 24. The method of any one of the preceding paragraphs, for treatment of TBI. 25. The method of any one of the preceding paragraphs, for treatment of concussion.

[0234] 26. The method of any one of the preceding paragraphs, wherein the treatment is of a neurological condition involved with elevated glutamate levels.

[0235] 27. The method of any one of the preceding paragraphs, wherein the treatment is of a neurological condition other than neurological damage and is selected from the groupconsisting of migraine, epilepsy amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and Huntington’s disease.

[0236] 28. The method of any one of the preceding paragraphs, comprising any one of intravenous administration, intramuscular administration and subcutaneous administration of the combination.

[0237] 29. The method of any one of the preceding paragraphs, wherein the first administration (be it an early administration within a time window of up to 4 hours or a late administration at a time window starting more than 4 hours from trauma or injury), comprises intravenous administration of the combination and any additional administration is selected from the group consisting of intravenous, intramuscular and subcutaneous administration.

[0238] 30. Use of a combination for treatment of a subject in need thereof, the combination comprising the following components: (i) a first transaminase (ii) a second transaminase that is different from the first transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions for essentially simultaneous administrations.

[0239] 31. The use of paragraph 30, wherein the first transaminase and the second transaminase are selected from glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

[0240] 32. The use of paragraphs 30 or 31, wherein the first substrate and the second substrate are selected from pyruvate (Pyr) and oxaloacetate (OxAc).

[0241] 33. The use of any one of paragraphs 30 to 32, wherein the co-factor is pyridoxal phosphate (PLP).

[0242] 34. The use of any one of paragraphs 30 to 33, wherein the treatment comprises administration of the combination comprising GPT as the first transaminase, GOT as the second transaminase, Pyr as the first substrate to the first transaminase, OxAc as the second substrate to the second transaminase, and PLP as the co-factor.35. The use of any one of paragraphs 30 to 34, wherein the treatment comprises administrations of the first substrate separately from the first transaminase and / or the second substrate separately from the second transaminase.

[0243] 36. The use of any one of paragraphs 30 to 35, wherein the treatment comprises administration of the first transaminase in a pharmaceutical composition with the first substrate and the second transaminase in another pharmaceutical composition, with the second substrate of the second transaminase.

[0244] 37. The use of any one of paragraphs 30 to 35, wherein the treatment comprises administration of a single pharmaceutical composition comprising the first transaminase, the first substrate to the first transaminase, the second transaminase and the second substrate of the second transaminase, and the transaminase co-factor.

[0245] 38. The use of any one of paragraphs 30 to 37, wherein the treatment is of neurological damage caused by trauma or injury.

[0246] 39. The use of paragraph 38, wherein the treatment comprises a first administration of the combination at a first time point after the trauma or injury, the first time point being within a first treatment window comprising any time point from the trauma / injury and up to 4 hours post trauma.

[0247] 40. The use of paragraph 39, comprising at least one additional administration of the combination after the first treatment window (but typically not more than 24 hours post injury / trauma), the combination of the at least one additional administration being formulated the same or different from the combination of the first administration. 41. The use of paragraph 40, comprising two or more of the at least one additional administration at respectively two or more additional time points after the first treatment window.

[0248] 42. The use of paragraph 41, wherein at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma or injury.

[0249] 43. The use of any one of paragraphs 30 to 38, comprising a first administration of the combination at a time point within a late treatment window that is greater than 4 hours post injury or trauma, typically more than 6 hours post injury or trauma.44. The use of paragraph 43, wherein the late treatment window is up to 36 hours post injury or trauma, i.e. the first administration is between 6 and about 36 hours post trauma or injury.

[0250] 45. The use of paragraph 43 or 44, wherein the first administration, being within the late treatment window, is 6-12 hours post injury or trauma.

[0251] 46. The use of any one of paragraphs 43 to 45, comprising at least one additional administration of the combination subsequent to the first (late time window) administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration. 47. The use of any one of paragraphs 30 to 46, when having two or more additional administrations to the first administration, the two or more additional administrations are daily administrations of the combination.

[0252] 48. The use of any one of paragraphs 30 to 47, wherein the treatment provides an effect that is greater, as defined herein, than the effect achieved with treatment with a single transaminase, a single substrate to the transaminase and a transaminase co-factor (determined by acceptable parameters as described herein).

[0253] 49. The use of paragraph 48, wherein the treatment provides an effect that is greater, as defined herein, than the effect achieved when the treatment is with glutamate-scavenging agent comprising GOT, OxAc and PLP (determined by acceptable parameters as described herein).

[0254] 50. The use of any one of paragraphs 30 to 49, providing a change in any one of the following parameters: reduction of glutamate level in blood and / or cerebrospinal fluid (CSF), reduction in edema size and / or lesion size, reduction of at least one inflammatory marker, reduction of astrocytic gliosis, reduction of scarring, improvement in cognitive and / or motor behavior, the change being determined in comparison to the parameter before treatment with the combination or in comparison to an acceptable reference level.

[0255] 51. The use of any one of paragraphs 30 to 50, wherein the treatment is of a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, haemorrhagic braininjury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition. 52. The use of paragraph 51, for treatment of SCI.

[0256] 53. The use of paragraph 51, for treatment of TBI.

[0257] 54. The use of paragraph 51, for treatment of concussion.

[0258] 55. The use of any one of paragraphs 30 to 54, wherein treatment is of a neurological condition involved with elevated glutamate levels.

[0259] 56. The use of paragraph 55, wherein the neurological condition is other than one resulting from injury and trauma and is selected from the group consisting of migraine, epilepsy amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and Huntington’s disease.

[0260] 57. The use of any one of paragraphs 30 to 56, wherein the first administration (either according to an early administration regime within an early time window of up to 4 hours or according to a late administration regime within a late time window of more than 4 hours and less than 36 hours, as defined herein) is by any one of intravenous administration, intramuscular administration and subcutaneous administration.

[0261] 58. The use of any one of paragraphs 31 to 57, wherein the first administration (either according to an early administration regime within an early time window of up to 4 hours or according to a late administration regime within a late time window of more than 4 hours and less than 36 hours, as defined herein) is by intravenous administration and the at least one additional administration is selected from the group consisting of intravenous, intramuscular and subcutaneous administration.

[0262] 59. A combination for use in treatment of a subject in need thereof, the combination comprising the following components: (i) a first transaminase (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined composition or as separate compositions for simultaneous or sequential administration.60. The combination for use of paragraph 59, wherein the first and second transaminase are different and are selected from the group consisting of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

[0263] 61. The combination for use of paragraphs 59 or 60, wherein the first substrate and second substrate are selected from the group consisting of pyruvate (Pyr) and oxaloacetate (OxAc).

[0264] 62. The combination for use of any one of paragraphs 59 to 61, wherein the cofactor is pyridoxal phosphate (PLP).

[0265] 63. The combination for use of any one of paragraphs 59 to 62, comprising administration of a combination comprising GPT as the first transaminase, GOT as the second transaminase, Pyr as the first substrate to the first transaminase, OxAc as the second substrate to the second transaminase, and PLP as the co-factor.

[0266] 64. The combination for use of any one of paragraphs 59 to 63, wherein the treatment comprises administrations of the first substrate separately from the first transaminase and / or the second substrate separately from the second transaminase. 65. The combination for use of any one of paragraphs 59 to 64, wherein the treatment comprises administration of the first transaminase in a same pharmaceutical composition with the first substrate and the second transaminase in a same pharmaceutical composition with the second substrate of the second transaminase. 66. The combination for use of any one of paragraphs 59 to 64, wherein the treatment comprises administration of a single pharmaceutical composition comprising the first transaminase, the first substrate to the first transaminase, the second transaminase and the second substrate of the second transaminase.

[0267] 67. The combination for use of any one of paragraphs 59 to 66, wherein treatment is of neurological damage caused by trauma or injury.

[0268] 68. The combination for use of paragraph 67, wherein the treatment comprises a first administration of the combination at a first time point after the trauma, the first time point being within a first (early) treatment window comprising any time point from the trauma or injury and up to 4 hours post trauma or injury.69. The combination for use of paragraph 68, comprising a first administration of the combination at the first time point and at least one additional administration of the combination after the first treatment window, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration.

[0269] 70. The combination for use of paragraph 69, comprising two or more of the at least one additional administration of the combination at respectively two or more additional time points after the first treatment window.

[0270] 71. The combination for use of paragraph 70, wherein at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma or injury.

[0271] 72. The combination for use of paragraph 67, comprising a first administration of the combination at a time point within a late treatment window that is greater than 4 hours post injury or trauma.

[0272] 73. The combination for use of paragraph 72, wherein the late treatment window is up to 36 hours post injury or trauma.

[0273] 74. The combination for use of paragraph 72 or 73, wherein the first administration according to the late treatment window (as part of the late treatment regime) is 6-12 hours post injury or trauma.

[0274] 75. The combination for use of any one of paragraphs 72 to 74, comprising at least one additional administration of the combination subsequent to the first administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration.

[0275] 76. The combination for use of paragraph 75, comprises two or more of the at least one additional administration.

[0276] 77. The combination for use of any one of paragraphs 59 to 76,, wherein when including two or more additional administrations, the two or more additional administrations (following the first administration, irrespective of whether the first administration is according to the early or late treatment regime) are daily administrations of the combination.78. The combination for use of any one of paragraphs 59 to 77, wherein the treatment provides an effect that is greater, as defined herein, than the effect achieved with treatment with a single transaminase, a single substrate to the transaminase and a transaminase co-factor (determined by acceptable parameters as described herein). 79. The combination for use of paragraph 78, wherein the treatment provides an effect that is greater, as defined herein, than the effect achieved when the treatment is with GOT, OxAc and PLP (determined by acceptable parameters as described herein).

[0277] 80. The combination for use of any one of paragraphs 59 to 79, providing a change in any one of the following parameters: reduction of glutamate level in blood and / or cerebrospinal fluid (CSF), reduction in edema size and / or lesion size, reduction of at least one inflammatory marker, reduction of astrocytic gliosis, reduction of scarring, improvement in cognitive and / or motor behavior, the change being determined in comparison to the parameter before treatment.

[0278] 81. The combination for use of any one of paragraphs 59 to 80, for treatment of a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, haemorrhagic brain injury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition. 82. The combination for use of paragraph 81, for treatment of SCI.

[0279] 83. The combination for use of paragraph 81, for treatment of TBI.

[0280] 84. The combination for use of paragraph 81, for treatment of concussion.

[0281] 85. The combination for use of any one of paragraphs 59 to 84, wherein the first administration (be it according to the early first administration regime or the late first administration regime) is by any one of intravenous administration, intramuscular administration and subcutaneous administration.

[0282] 86. The combination for use of any one of paragraphs 59 to 66, wherein the first administration (be it according to the early first administration regime or the late first administration regime) comprises intravenous administration and the at least oneadditional administration is selected from the group consisting of intravenous, intramuscular and subcutaneous administration.

[0283] 87. The combination for use of any one of paragraphs 59 to 86, wherein the treatment is of a neurological condition involved with elevated glutamate levels.

[0284] 88. The combination for use of paragraph 87, wherein the neurological condition is other than one resulting from trauma or injury and is selected from the group consisting of migraine, epilepsy amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and Huntington’s disease.

[0285] 89. The combination for use of paragraph 88, wherein administration is by oral delivery.

[0286] 90. A pharmaceutical product comprising a combination of components:

[0287] (i) a first transaminase

[0288] (ii) a second transaminase,

[0289] (iii) a first substrate to the first transaminase

[0290] (iv) a second substrate to the second transaminase, and

[0291] (v) a transaminase co-factor,

[0292] wherein the components are formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions. 91. The pharmaceutical product of paragraph 90, wherein the first transaminase and the second transaminase are different and are selected from the group consisting of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

[0293] 92. The pharmaceutical product of paragraph 90 or 91, wherein the first substrate and the second substrate are selected from the group consisting of pyruvate (Pyr) and oxaloacetate (OxAc).

[0294] 93. The pharmaceutical product of any one of paragraphs 90 to 92, wherein the cofactor is pyridoxal phosphate (PLP).

[0295] 94. The pharmaceutical product of any one of paragraphs 90 to 93, wherein the first transaminase is GPT, the second transaminase is GOT, the first substrate to the firsttransaminase is Pyr, the second substrate to the second transaminase is OxAc and the transaminase co-factor is PLP.

[0296] 95. The pharmaceutical product of any one of paragraphs 90 to 94, wherein when the components are formulated for administration as separate pharmaceutical compositions, the first substrate is formulated separately from the first transaminase and / or the second substrate is formulated separately from the second transaminase. 96. The pharmaceutical product of any one of paragraphs 90 to 94, wherein when the components are formulated for administration as separate pharmaceutical compositions, the first transaminase in a same pharmaceutical composition with the first substrate and the second transaminase in a same pharmaceutical composition with the second substrate of the second transaminase.

[0297] 97. The pharmaceutical product of any one of paragraphs 90 to 96, wherein the components are formulated for administration as a single combined pharmaceutical composition comprising the first transaminase, the first substrate to the first transaminase, the second transaminase and the second substrate of the second transaminase and the transaminase co-factor.

[0298] 98. The pharmaceutical product of any one of paragraphs 90 to 97, comprising instructions for use of the combination of components in treating neurological condition.

[0299] 99. The pharmaceutical product of any one of paragraphs 90 to 98, comprising instructions for use in treating neurological condition caused by trauma or injury. 100. The pharmaceutical product of paragraph 98 or 99, wherein the instructions specify a first administration of the combination at a first time point after the trauma or injury, the first time point being within a first treatment window (referred to as the early first administration) comprising any time point of up to 4 hours post trauma or injury.

[0300] 101. The pharmaceutical product of paragraph 100, wherein the instructions specify at least one additional administration of the combination after the first treatment window, wherein the combination for the at least one additional administration is formulated the same or differently from the combination of the first administration.102. The pharmaceutical product of paragraph 100, wherein the instructions specify two or more additional administrations of the combination at respectively two or more additional time points after the first treatment window.

[0301] 103. The pharmaceutical product of paragraph 102, wherein the instructions specify that at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma.

[0302] 104. The pharmaceutical product of paragraph 98 or 101, wherein the instructions comprise a first administration of the combination at a time point within a late treatment window that is greater than 4 hours post injury or trauma (referred to as the late first administration).

[0303] 105. The pharmaceutical product of paragraph 104, wherein the late treatment window is up to 36 hours post injury or trauma.

[0304] 106. The pharmaceutical product of paragraph 104 or 105, wherein the first administration (according to the late first administration regime) is 6-12 hours post injury or trauma.

[0305] 107. The pharmaceutical product of any one of paragraphs 100 to 106, wherein the instructions comprise at least one additional administration of the combination subsequent to the first administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration (the first administration can be within the early time window or late time window).

[0306] 108. The pharmaceutical product of paragraph 107, wherein the instructions comprise two or more of the at least one additional administration.

[0307] 109. The pharmaceutical product of any one of paragraphs 100 to 108, wherein when comprising two or more additional administration following the first administration, the two or more additional administrations are daily administrations of the combination.

[0308] 110. The pharmaceutical product of any one of paragraphs 90 to 109, for treating a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, haemorrhagic braininjury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition. 111. The pharmaceutical product of paragraph 110, for treatment of SCI.

[0309] 112. The pharmaceutical product of paragraph 110, for treatment of TBI.

[0310] 113. The pharmaceutical product of paragraph 110, for treatment of concussion. 114. The pharmaceutical product of any one of paragraphs 90 to 99, wherein the treatment is of a neurological condition involved with elevated glutamate levels.

[0311] 115. The pharmaceutical product of any one of paragraphs 90 to 114, wherein the first administration is by any one of intravenous administration, intramuscular administration and subcutaneous administration.

[0312] 116. The pharmaceutical product of any one of paragraphs 90 to 115, wherein the first administration comprises intravenous administration and any additional administration is selected from the group consisting of intravenous, intramuscular and subcutaneous administration.

[0313] 117. The pharmaceutical product of any one of paragraphs 90 to 116, wherein each administration is formulated for any one of intravenous administration, intramuscular administration and subcutaneous administration.

[0314] 118. The pharmaceutical product of paragraph 98, wherein the neurological condition is other than neurological damage caused by trauma or injury and is selected from the group consisting of migraine, epilepsy amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and Huntington’s disease.

[0315] 119. The pharmaceutical product of paragraph 118, wherein the components are formulated for oral administration.

[0316] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0317] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some casesup to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

[0318] The indefinite articles “a” and “an” as used herein in the description and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one". It must be noted that, as used in this description and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0319] The clause “and / or” as used herein in the description and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified.

[0320] As used herein in the description and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of or “exactly one of” or, when used in the claims, “consisting of” will refer to the inclusion of exactly one element of a number or list of elements.

[0321] As used herein in the specification and in the claims, the phrase “at least one” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0322] Throughout this description (including the Examples) and claims which follow, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", “involving”, "holding", "composed of and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to". The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0323] It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and " ranging / ranges from" a first indicate number "to" a second indicate number are usedherein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0324] It is appreciated that certain features of the presently disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0325] Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

[0326] Disclosed and described, it is to be understood that the presently disclosed subject matter is not limited to the particular examples, process steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the presently disclosed subject matter will be limited only by the appended claims and equivalents thereof.

[0327] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the presently disclosed subject matter.DESCRIPTION OF NON-LIMITING EXAMPLES

[0328] Example 1 - Glutamate levels in plasma

[0329] Materials & Methods

[0330] rGOTl - His-tagged recombinant rat glutamate-oxaloacetate transaminase (rGOTl) cDNA was cloned from the human hepatoma cell line hepG2, expressed in Escherichia coli, and purified by Ni-agarose chromatography [Goldshmit Y, et al., J Neurotrauma. 2018;35(21):2581-2590], The rat GOT1 protein has the amino acid sequence as denoted by SEQ ID NO: 6.

[0331] rGPTl - mouse rGPTl enzyme was purchased from Abeam (ab222953) and has the amino acid sequence as denoted by SEQ ID NO: 7.

[0332] The activity of the enzymes was determined using a Reflotron automatic analyzer (Roche).

[0333] OxAc, andPLP - were purchased from Sigma

[0334] Blood plasma - Blood was collected from the submandibular vein into tubes containing 2 pL heparin. Tubes were centrifuged at +4°C and 16000g for 5 minutes; next, the plasma was separated and used immediately or stored at -80°C.

[0335] Preparation of rGOTl and OxAc + PLP Reference treatment:

[0336] The rat rGOTl complementary DNA (cDNA) was cloned into the pET28 vector and transformed into Escherichia coli BL21(DE3) bacteria; protein expression was induced by the addition of isopropyl b-D-1 -thiogalactopyranoside (IPTG; 0.2 mM). Next, the recombinant His-tagged rGOTl protein was purified by Ni-agarose chromatography and concentrated using a VivaSpin20 centrifuge. The quality and purity of the purified rGOTl protein were assessed on SDS-PAGE, quantified using a spectrophotometer, and the activity was measured using a Reflotron automatic analyzer (Roche).

[0337] The mice were injected I. V or IP according to the experiment’s parameters with 30pg (50mg / ml) of rGOTl in combination with 0.03M OxAc (Sigma), pH of OxAc was adjusted to 6.5 using 5M NaOH. The total volume injected into each mouse was 200ul of saline containing rGOTl with OxAc (0.03M) and PLP (Sigma, 0.008pM).Preparation of GPT + Pyruvate + PLP Reference treatment:

[0338] rGPTl enzyme was processed according to the manufacturer's protocol, and the activity of the rGPTl enzyme activity was determined using a Reflotron automatic analyzer (Roche). 50pg rGPTl (30mg / ml concentration) in combination with 0.03M Pyruvate and the pH was adjusted to 5.5 using 5M NaOH. The total volume injected to each mouse was 200ul of saline containing rGPTl with pyruvate (0.03M) and PLP (0.008pM).

[0339] Preparation ofrGOTl+OxAc+rGPTl+Pyruvate+PLP (combined BGS) treatment:

[0340] The preparation of the combined BGS formulation for “combined” treatment was done by combining both individual treatments as described above. First, the substrates Pyruvate and OxAc were combined at 0.03M, adjusting pH to 5.5 using 5M NaOH. And then the rGOTl and rGPT enzymes were added. The total volume injected to each mouse was 200pl of saline containing rGPTl + rGOTl with pyruvate and OxAc at 0.03M and PLP (Sigma) 0.008pM.

[0341] In vitro experiment

[0342] For in-vitro experiments, glutamate was added to the extracted blood at a concentration of 200 pM in order to emulate the rise in glutamate levels that occurs after spinal cord injury. The blood with glutamate and heparin was allocated to tubes with 300pl per tube and divided into three blood glutamate scavenging "BGS") treatment groups:

[0343] rGOTl + OxAc rGOT1 (4.5μg) + OxAc (0.03M) +PLP (0.008pM)

[0344] rGPTl + Pyruvate: GPT (7.47pg) + Pyr (0.03M) + PLP(0.008pM)

[0345] Combined BGS (cBGS): rGOT1 (4.5μg) + OxAc (0.03M) + GPT (7.47pg) + Pyr (0.03M) +PLP (0.008pM).

[0346] Levels of glutamate were measured up to 90 minutes by HPLC. The glutamate level was analyzed with precolumn derivatization with o-phthalaldehyde (OPA) reagent and separated by reverse-phase HPLC with a scanning fluorescence detector. The excitation and emission wavelengths were 350 and 460 nm, respectively. Chromatography was performed using the UltiMate 3000 LC system (Thermo Scientific). The amino acid standard mix (Sigma-Aldrich) or samples were pre-columnderivatized with an OP A reagent solution. The derivatization reagent was 5.0 mg of OPA dissolved in 100 pl of methanol and diluted with 900 pl of 0.4 M borate buffer (pH 9.5) and 5 pl of P-mercaptoethanol.

[0347] For blood plasma samples, an additional step was added; the plasma was precipitated with the use of sulfosalicylic acid (SSA) at a ratio of one part plasma to two parts SSA (1:2). For derivatization of amino acids, standard or a CSF / plasma sample was mixed with 5 pl of OPA derivatization reagent in the autosampler. All chromatographic separations were performed on a Hypersyl Gold 5U column (4.6 mm x 250 mm, 2.5 pl; Thermo Scientific). The amino acid concentration was determined using the peak area and the external standard method.

[0348] Results

[0349] Reference is made to Figure 1 showing time-course of glutamate levels in blood extracted from naive mice, in vitro, after the blood was treated with one of the above treatment groups.

[0350] Figure 1 shows that the combined BGS treatment (Group III, cBGS) demonstrated the lowest levels of glutamate at all time points. Additionally, the cBGS treatment significantly reduced glutamate levels compared to each of the other Reference treatment groups, namely, Reference treatment Group I (rGPTl + Pyr + PLP) and Reference treatment Group II (rGOTl+OxAc+PLP), at all-time intervals.

[0351] Example 2 - Glutamate levels in spinal cord injury (SCI) model

[0352] The purpose of the experiment was to characterize the acute phase of SCI and to prove that cBGS successfully reduces CSF glutamate levels after SCI.

[0353] (A) Spinal Cord Injury moderate / severe Compression Model:

[0354] Mice were anesthetized by intraperitoneal injections (i.p.) of ketamine (60 mg / kg) and xylazine (10 mg / kg) (at least 5 mice per group, C57BL / 6 mice). To examine whether the proposed combined cBGS treatment is feasible for compression injury, a blood-vessel clip compression model was used [AS Medizintechnik GmbH; Germany], applying a closing force of 10 gr, 1mm width. The laminal arch of the vertebrae at the tenth thoracic level was removed, and the exposed dura mater was subjected to compression by a blood-vessel clip for 5 sec. In the Sham group only thelaminal arch of the vertebrae at the tenth thoracic level was removed without spinal cord injury.

[0355] (B) Spinal Cord Injury Contusion Model:

[0356] Mice (C57BL / 6 mice, 5 per group) were anesthetized by intraperitoneal injections of ketamine (60 mg / kg) and xylazine (10 mg / kg). The laminal arch of the vertebrae at the tenth thoracic level was removed, and the exposed dura mater was subjected to a Moderate Contusive SCI: speed 1msec, depth 0.5mm, time 0.5s, or to a Severe Contusive SCI: speed 1msec, depth 1mm, time 0.5s, using a commercially available SCI device (Precise Impactor, Scintica Instrumentation Inc.).

[0357] (C) Cerebrospinal fluid (CSF sampling and analysis:

[0358] For this experiment, moderate / severe compression SCI was induced in a separate group of C57BL / 6 mice. CSF was extracted from each mouse under Isoflurane anesthesia by glass capillary from the cisterna magna at 28h post-injury and four hours after cBGS.

[0359] Treatment groups

[0360] Sham Group: mice that were anesthetized, and the skin was cut without inducing Spinal cord injury, and injected with vehicle (0.9% Saline).

[0361] Control Group: mice that were induced with Spinal cord injury followed by vehicle only (0.9% Saline) injection.

[0362] Naive group healthy mice.

[0363] In the SCI compression model (A above), the following treatment groups were tested:

[0364] Naive, Sham, and Control groups

[0365] Reference treatment group: rGOTl / OxAc / PLP

[0366] Reference treatment group: rGPTl / Pyr / PLP

[0367] Combined treatment “cBGS” group: rGOTl / OxAc / rGPTl / Pyr / PLP

[0368] The treatment included a first i.v. administration 1 or 4 hours after the injury. In some experiments, additional treatments were given, these included five consecutivedaily administrations. While the first injection was administrated intravenously, it was, in these cases, followed by four intraperitoneal injections.

[0369] In the SCI contusion model (B, above) the following treatments were tested: Naive, Sham, and Control groups

[0370] Combined treatment rGOTl / OxAc / rGPTl / Pyr / PLP

[0371] The treatment was administered 1 hour after the injury, intravenously (i.v), followed by four intraperitoneal injections (i.p).

[0372] In the CSF sampling (C above) the following treatment groups were tested: Naive, Sham and Control groups

[0373] Reference treatment group: rGOTl / OxAc / PLP

[0374] Combined cBGS treatment group: rGOTl / OxAc / rGPT / Pyr / PLP

[0375] The treatment was administered 1 or 4 hours after the injury, for one or two consecutive days on a daily basis, while the first injection was administrated intravenously, followed by four intraperitoneal injections.

[0376] Analysis

[0377] Glutamate levels were analyzed by HPLC with Pre-Column Derivatization with o - phthalaldehyde (OP A) reagent and separated by reversed-phase HPLC with a scanning fluorescence detector (HPLC described above).

[0378] Results

[0379] Moderate / severe compression SCI or contusion SCI were performed according to procedures (A) or (B) above, and each mouse received one of the treatments specified above, with the first injection being administrated, i.v, 1 hour or 4 hours post injury and in some cases, with the additional 4 injections being administered i.p on a daily basis, the first additional injection being 24 hours after the first one.

[0380] Figures 2A-2C show the effect of the combined treatment (cBGS) vs. any one of the control groups (Sham, control or naive mice) as well as vs. the Reference treatment with rGOTl+OxAc+PLP. Results are presented as mean ± SEM.Specifically, Figure 2A shows CSF Glutamate levels one day post-compression spinal cord injury and after two injections of treatment, while the first one was administered one hour after the injury. The results show that the SCI induced a significant elevation in CSF glutamate levels compared to the naive uninjured mice and Sham (mice that went under the surgery without injury). cBGS treatment significantly reduced glutamate levels, balancing Glu almost to normal levels.

[0381] Figure 2B shows CSF Glutamate levels two days post-compression spinal cord injury and after three injections of treatments, while the first one was administrated four hours after the injury. Similar to Figure 2A, the results in Figure 2B show that the SCI induced a significant elevation in CSF glutamate levels compared to the Sham uninjured mice. The one-day or two-day Reference treatment with rGOTl / OxAc / PLP did not significantly decrease the glutamate level, while, on the contrary, the 2-inj ection Combined treatment reduced it almost up to the normal level and below the level in the Sham group, which demonstrated a slightly elevated level of glutamate.

[0382] Figure 2C shows CSF Glutamate levels one day post-contusion spinal cord injury and after two injections of treatments, while the first one was administered one hour after the injury, and an additional injection 24 hours after the first injection. The results show that the SCI induced a significant elevation in CSF glutamate levels compared to the Sham uninjured mice. The two injections treatment with the Reference group: rGOTl / OxAc / PLP did not significantly decrease the glutamate level, while on the contrary, the combined cBGS treatment reduced it almost up to the normal level and below the level in the sham group that demonstrated a slightly elevated level of glutamate.

[0383] To summarize, the results show the reduction of glutamate levels in CSF in the cBGS treatment group. The reduction is statistically significantly greater as compared to the control, Sham or Reference treatment group (treatment with only rGOTl+OxAc+PLP).

[0384] Example 3 - Axon Characterization in Spinal Cord Injury Model

[0385] The purpose of the experiment was to examine whether the combined, cBGS treatment provides a neuroprotective effect following injury.Methods

[0386] Moderate / severe compression SCI was induced on mice, which were subsequently treated with the following treatment groups

[0387] cBGS formulation'. rGOTl (30pg) + OxAc (0.03M) + rGPTl (50pg) + Pyr (0.03M) + PLP (0.008pM) per one mouse

[0388] Reference formulation'. rGOT1 (30µg) + OxAc (0.03M) + PLP (0.008pM) in 200 pl per one mouse.

[0389] The treatment groups included:

[0390] cBGS (#1): A single i.v injection 4 hours post injury (referred to as “1 injection Combined”, n=8)

[0391] cBGS (#5): A total of 5 injections, including a first i.v. injection 4 hours post-injury followed by 4 daily i.p. injections of the same composition (referred to as “5 days Combined", n=11)

[0392] Reference rGOTl + OxA c +PLP — A total of 5 injections, including a first i.v. injection 4 hours post-injury followed by 4 daily i.p. injections of the same composition (referred to as “5 days rGOTl OxAc", n=12)

[0393] - Control - A total of 5 injections, including a first i.v. injection 4 hours postinjury followed by 4 daily i.p. injections of the 0.9% Saline (n=11) After seven weeks, the mice were sacrificed, and the injured site was analyzed by determining number of axons

[0394] Spinal cord tissue sections (at least 5 mice per group) were frozen in OCT. The CRYOSTAT was sectioned at 60 pm, and the sections were stained with primary and secondary Abs.

[0395] Determining the number of axons:

[0396] Sections (at least 5 sections from each mouse) were imaged by fluorescence microscopy using an Axioplan Z1 (Zeiss) epifluorescence microscope. Photomicrographs (1300 x 1030 dpi) were obtained with 10x and 20x Plan-Neofluar (Zeiss, Germany) objectives, and acquired using an AxioCam (Zeiss, Germany) digital camera using AxioVision software (v. 4.4; Zeiss, Germany). Images were edited and colored usingAdobe Photoshop 11, and density analyses were completed using ImageJ. Axons crossing the lesion site were counted manually in a double-blind way. NeuN was also visualized using the SP8 LIGHTNING confocal microscope (Leica Microsystems).

[0397] Immunohistochemistry:

[0398] Cryostat longitudinal floating sections (60 pm) of fixed frozen tissue were stained using standard immunohistochemistry. A series of 60 pm-thick longitudinal sections were used for each antibody at the lesion site (10 slides from 6 animals from each group were analyzed). Primary antibodies: rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, Dako); rabbit anti-Ibal (1:500; Abeam); mouse anti-NeuN (1:1000; Millipore); rabbit anti-myelin basic protein (MBP; 1:500, Abeam); and Rabbit anti-Synaptophysin (1:1000, Abeam). Secondary antibodies: Alexa Fluor 488, 633 or 568; 1:1000 (Invitrogen). Nuclei were visualized with DAPI (Sigma). The immunofluorescence intensity and density were measured for GFAP, Ibal, MBP, and Synaptophysin. For each measurement, sections were taken at 200-mm intervals. All measurements were performed using Image J. Sections were imaged by fluorescence microscopy using an Olympus IX83 fluorescent microscope using Cell Sens Dimension software.

[0399] Results

[0400] Figure 3 shows axonal survival seven weeks following moderate / severe Spinal cord injury (SPI) and illustrates how treatment of the SCI mice with additional administrations of cBGS, 5 consecutive days after injury, significantly increased axonal survival compared to control as well as compared to the same schedule of treatment with only rGOTl+OxAc+PLP.

[0401] An additional long-term experiment was conducted to further investigate the neuroprotective effects of the cBGS treatment. The microenvironment at the chronicstage lesion site, known for its significant influence on functional recovery, was examined using the moderate / severe compression SCI model, comparing the 5-day combined treatment (as detailed above) to the control group. The neuronal rescue and lesion size seven weeks post moderate / severe compression SCI were analyzed.Figures 4A-4C show neuronal rescue and lesion size with the combined treatment, following moderate / severe compression SCI, seven weeks post moderate / severe compression SCI.

[0402] Specifically, as shown in Figures 4A, the density of neurons shown by NeuN staining around the lesion site was statistically significantly higher in the cBGS-treated group, i.e. that received the combined treatment (referred to in the figure as “GOT + GPT”) as compared to the control (“CON”) group. It is noted that a comparison with only rGOTl+OxAc+PLP is provided in Figs. 6 and 7.

[0403] Further, in order to assess the functionality of these neurons, the number of synapses density was measured exclusively around neurons that were NeuN positive. As shown in Figure 4B, the combined cBGS treatment (referred to as “GOT+GPT”) exhibited a statistically significantly greater amount of synaptophysin around these neurons in comparison to the control group.

[0404] Finally, as shown in Figure 4C, the total area of the lesion site was measured, demonstrating the ability of combined treatment (referred to as “GOT+GPT”) to significantly lower the total area of the lesion site (control n=8; 5-day combined n=5).

[0405] Example 4 - Characterization in severe contusion spinal cord injury model Based on the promising results in the moderate-severe SCI model, the effect of the combined treatment was tested in a more devasting injury, which is severe contusion SCI.

[0406] Methods

[0407] To visualize all motor and sensory neurons and their projection, transgenic mice Tg (Thyl-EYFP) were used. The transgenic mice enable examination of neuronal and axonal degeneration / regeneration at the lesion area after BGS treatment. All procedures were approved by the Tel-Aviv University Animal Ethics Committee in accordance with the requirements of the National Health and Medical Research Council of Israel.

[0408] Spinal cord injury contusion severe model:

[0409] Thyl-YFP adult mice (treated group n=7, control untreated n=5) were anesthetized by intraperitoneal injections of ketamine (100 mg / kg) and xylazine (10mg / kg). The spinal cord was exposed at the low-thoracic to high-lumbar area. The mice were then positioned on an impactor machine (Precise Impactor, Scintica Instrumentation Inc). Each mouse was subjected to an impactor hit at T12 with a speed of 2.5m / s at a depth of 0.8mm and a dwell time of 0.5 seconds.

[0410] Treatment groups

[0411] The mice were treated as follows:

[0412] cBGS: single i.v. injection, 1 hour post injury with a combination comprising rGOTl (30pg) + OxAc (0.03M) + rGPTl (50pg) + Pyr (0.03M) +PLP (0.008pM), followed by 4 additional daily i.p. injections, the first additional injection being 24 hours after the first injection.

[0413] Control: A total of 5 injections, including a first i.v. injection 1-hour post-injury followed by 4 daily i.p. injections of the 0.9% Saline.

[0414] GFAP, Ibal, axon crossing, and BMS analysis

[0415] Immunohistochemistry and fluorescent microscopy of spinal cord tissue

[0416] Spinal cord tissue sections were frozen in OCT and CRYOSTAT sectioned at 60 pm.

[0417] Primary antibodies were:

[0418] Rabbit anti-glial fibrillary acidic protein (GFAP). Dako. 1: 1000

[0419] - Rabbit anti-ionized calcium binding adaptor molecule 1 (IBA1). Abeam. 1:500 Mouse anti-glial fibrillary acidic protein (GFAP). Abeam. 1:1000 Secondary antibodies were Alexa Fluor 488 or 568 (1: 1000, Invitrogen) and DAPI (6-diamidino-2-phenylindole, Sigma) to visualize nuclei, and distinguish the lesion borders. Sections were also used for axonal counting across the lesion site.

[0420] Sections were imaged by fluorescence microscopy using an Axioplan Z1 (Zeiss) epifluorescence microscope. Photomicrographs (1300 x 1030 dpi) were obtained with 10x and 20x Plan-Neofluar (Zeiss, Germany) objectives, and acquired using an AxioCam (Zeiss, Germany) digital camera using AxioVision software (v. 4.4; Zeiss, Germany). Images were edited and colored using Adobe Photoshop 11, and density analyses werecompleted using ImageJ. Axons crossing the lesion site were counted manually in a doubleblind way.

[0421] Basso Mouse Scale:

[0422] Evaluated for 3 minutes using the Basso Mouse Scale (BMS). Mice were tested for hindlimbs functional deficits up to 3 weeks after SCI. Hindlimbs locomotor recovery was assessed in an open field using the BMS. This scale ranges from 0 (complete paralysis) to 9 (normal hindlimb movement). The performance of the left and right hindlimbs was averaged in order to obtain the BMS score.

[0423] Statistical analysis

[0424] All statistical analyses were conducted using GraphPad Prism, Version 9.0.0, for Windows. Significance was evaluated using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference test (Tukey's HSD) for multiple comparisons, a set to 5%) when data were normally distributed. Significance was evaluated using the non-parametric Kruskal -Wallis Test, followed by Dunn’s multiple comparisons with Bonferroni’s correction method, a set to 5%) when data were not normally distributed.

[0425] Results

[0426] The purpose of this experiment was to determine whether the combined cBGS treatment was effective in a more devastating injury, which is severe contusion SCI. The treatment was given 1 hour post-injury, and the inflammation levels were assessed one-week post-injury.

[0427] As shown in Figures 5A-5B, the combined cBGS treatment (referred to as “GOT+GPT”) induced a significant reduction in inflammatory response as demonstrated by lower levels of astrocyte activation (GFAP) and microglia / macrophage (Iba-1) density around the lesion site compared to the control untreated mice.

[0428] Moreover, as shown in Figure 5C, the combined cBGS treatment (“GOT+GPT”) exhibited significantly higher levels of axon survival across the lesion site compared to the control.In terms of functional motor recovery, an improvement was observed in BMS score over time in comparison to the control group, reaching statistical significance at 7 days time-point as shown in Figure 5D. The injured mice in the control group suffered from severe motor impairment and general deterioration.

[0429] Example 5 - Motor characterization in spinal cord injury compression model To assess the functional recovery of mice after SCI, two motor function tests, the Basso Mouse Scale (BMS) and the grid walk, were implemented.

[0430] Methods

[0431] Moderate / severe compression SCI was induced on mice as described above, which were subsequently treated 1 hour or 4 hours post injury as follows:

[0432] cBGS (#1): A single i.v. injection of a combination comprising rGOTl (30pg) + OxAc (0.03M) + rGPTl (50pg) + Pyr (0.03M) +PLP (0.008pM)

[0433] cBGS (#5) A total of 5 injections of the combination rGOTl (30pg) + OxAc (0.03M) + rGPTl (50pg) + Pyr (0.03M) +PLP (0.008pM), including a first i.v. injection 1 hour or 4 hours post injury, followed by 4 daily i.p. injections of the same combination. Reference rGOTl +OxAc+PLP - total of 5 injections of rGOTl (30pg) + OxAc (0.03M)+PLP (0.008pM), including a first i.v. injection 4 hours post-injury, followed by 4 daily i.p. injections.

[0434] Control - Injured mice treated with 5 injections of 0.9% Saline

[0435] The functional recovery of the mice in all groups was analyzed during the seven weeks post-injury at the indicated time points. Two motor function tests were used: the Basso Mouse Scale (BMS), and Grid Walk.

[0436] BMS test:

[0437] Evaluated for 3 minutes using the Basso Mouse Scale (BMS) as described above. Mice were tested for hindlimb functional deficits after SCI. Hindlimb locomotor recovery was assessed in an open field using the BMS scale. This scale ranges from 0, indicating complete paralysis, to 9, indicating normal movement of the hindlimb. The performance of the left and right hindlimbs was averaged in order to obtain the BMS score.Horizontal Grid Walking Test:

[0438] The mice were tested walking on a wire grid (1.2 × 1.2–cm grid spaces, 35 × 45–cm total area) after SCI. Each mouse was tested for 3 minutes of free walking on a grid, and the total number of steps was counted. When either hindlimb paw protruded entirely through the grid with all toes and heels, it was counted as a 1-foot miss. The results were expressed as the number of correct footsteps (total number of steps - missteps) as a percentage of the total number of steps taken for 3 minutes of free walking. The performance of the left and right hindlimbs was averaged in order to obtain the final Gridwalk score.

[0439] Regularity index, stride length, swing speed, and average run speed measured by Catwalk:

[0440] Gait measures were determined using the CatWalk XT 10.6 system (Noldus Technology) at the two-week time point. Each mouse was located on the platform and permitted to cross the walking path for at least 3 compliant runs. All measurements were analyzed separately for each hind paws.

[0441] Given that the majority of improvement observed in the long-term experiment occurred within the first two weeks, the mice were further tested at the two-week post-SCI timepoint using additional parameters, including regularity index, stride length, swing speed, and average run speed. These parameters are comparable to human measurements and relevant to human SCI.

[0442] Regularity index (RI) is defined as the exclusive use of normal step sequence patterns during uninterrupted locomotion. One way of measuring coordination is the regularity index, which relies heavily on the concept of regular step patterns. For fully coordinated locomotion, each paw is placed exactly once every four steps. The RI of the same mouse before injury was compared to the RI after the injury and vs. other groups.

[0443] Swing speed and stride length, both of which calculate the average between two consecutive steps, from when a foot is raised until it touches the ground again. Swing speed refers to the velocity at which this sequence happens, while stride length represents the average distance. Swing speed: Parameter calculated using swing and stride length, expressed in centimeters / second (cm / s). Stride length: Distance betweenpaw placement in two consecutive steps of the same paw, expressed in centimeters (cm). Average speed, which measures the average speed of three consecutive compliant runs and is representative of the general locomotion status. The Run Average Speed is the average speed of the animal's body during a run.

[0444] Results

[0445] As shown in Figures 6A-6C, in both motor behavioral assessments, all mice improved over time. However, the combined cBGS treatment groups exhibited a recovery of up to 80% compared to the untreated group, which exhibited only a 20% functional recovery. Notably, the 5-inj ection combined treatment proved to be the most effective among all the treatment options.

[0446] Further, as shown in Figure 6A, in the BMS score, when the treatment was administered one-hour post-injury, the combined cBGS treatment group exhibited significant functional improvement already at three days post-injury, while the rGOTl + OxAc + PLP treatment exhibited motor improvement from only one week.

[0447] Furthermore, as shown in Figure 6B-6C, when the combined cBGS treatment was administered four hours post injury, the 5-injection combined cBGS treatment showed significantly better / faster functional improvement as shown by the BMS score (Figure 6B) or the Grid walking (Figure 6C) compared to either a 1-injection of combined cBGS treatment or a 5-day treatment with rGOTl+OxAc.

[0448] To gain a deeper understanding of motor function rehabilitation, a gait analysis using a catwalk was performed two weeks post-SCI. Parameters analyzed included regularity index, stride length, swing speed, and average run speed, as they are similar in humans and relevant to human SCI.

[0449] As shown in Figures 7A-7D, in all the parameters, the 5-injection combined (cBGS) treatment group had significant motor improvement compared to the control / untreated mice, as well as in comparison to 5-injection treatment with only rGOTl +OxAc+PLP (Fig. 7D).

[0450] Further, the 5-day combined (cBGS) group regularity index reached almost 80% while none of the other groups reached a value higher than 50%, with the highest being 47% in the 5-day rGOTl + Ox Ac + PLP treated group (Figure 7A). The same patternwas observed for the swing speed and stride length where only the 5-day combined (cBGS) group reached a significant difference compared to the control mice (Figures 7B -7C).

[0451] Lastly, the run average speed in the 5-day combined cBGS) treatment group was significantly higher as compared to both the control group and the 5-day rGOTl + Ox Ac + PLP group (Figure 7D).

[0452] Example 6 - Spinal Cord Compression -late Treatment Window

[0453] Next, a belated therapeutic window following neurological injury (moderate / severe compression SCI) was examined. In this context, the belated treatment involved injection 8 hours post-injury.

[0454] Methods

[0455] Spinal cord injury moderate / severe compression model:

[0456] Adult (3-6 months) TgN(Thyl-EYFP) or their B16 / C57 WT littermates mice were used.

[0457] As described above, all mice (at least 8 mice per group) were anesthetized by intraperitoneal injections (i.p.) of ketamine (60 mg / kg) and xylazine (10 mg / kg). To induce compression injury, a well-established blood-vessel clip compression model (AS Medizintechnik GmbH; Germany), applying a closing force of 10 gr, 1mm width. The laminal arch of the vertebrae at the T10 level was removed, and the exposed dura mater was subjected to compression by a blood vessel clip for 5 sec.

[0458] Treatment groups:

[0459] Mice were randomly divided into several groups:

[0460] cBGS (comb 1inj): A single IV injection 8 hours post-injury. The injection was at a dose of 1 mg / kg of rGOTl + 0.03M OxAc combined with 1.5 mg / kg rGPTl + 0.03M Pyr + PLP 0.008pM.

[0461] cBGS (comb 5inj): total of 5 injections, including a first IV injection 8 hours post injury, followed by 4 daily i.p. injections. Each injection was at a dose of 1 mg / kg of rGOTl + 0.03M OxAc, combined with 1.5 mg / kg rGPTl + 0.03M Pyr + PLP 0.008 pM.rGOT1+OxAc +PLP (GOT lin )- A single IV inj ection 8 hours post injury. The injection is at a dose of rGOTl (1 mg / kg) + 0.03M OxAc + PLP (0.008pM).

[0462] rGOT1+OxAc +PLP (GOT 5inj)- total of 5 injections, including a first IV injection 8 hours post-injury, followed by 4 daily i.p. injections. Each injection was at a dose of 1 mg / kg of rGOTl + 0.03M OxAc + PLP (0.008pM).

[0463] rGPT1+Pyr+PLP(GPT 1inj) – A single IV injection 8 hours post injury. The injection is at a dose of: 1.5 mg / kg rGPTl + 0.03M Pyr + PLP (0.008pM).

[0464] Control-vehicle: mice were injected with the same regime with 200 pl 0.9% Saline.

[0465] BMS test, immunostaining analyses, myelin breakdown, synaptic degeneration, and neuronal death at the lesion site

[0466] BMS test, immunostaining analyses, myelin breakdown, synaptic degeneration, and neuronal death at the lesion site were all performed as described hereinabove.

[0467] Results

[0468] The functional recovery of the mice in all groups was analyzed up to three weeks post injury at the indicated time points.

[0469] As shown in Figure 8A, the motor hindlimb function, as measured by the BMS test was significantly improved with the 5-day combined cBGS (“comb 5inj”) treatment group compared to all other tested groups.

[0470] Moreover, as shown in Figure 8B, the motor function in the group treated with a single injection of the combined treatment (“comb linj”), 8 hours after the primary insult, was significantly improved over that treatment with each component separately.

[0471] It was thus concluded that the combined cBGS treatment significantly improves functional recovery after spinal cord injury in Extended Treatment Window Further, the expression of GFAP astrocytic marker expression was examined in the 8-hour first injection window in both the 5-day combined BGS-treated mice and vehicle-control groups.

[0472] As shown in Figures 9A-9B, the combined cBGS treatment, including a first injection 8 hours post-injury, reduces astrocyte reactivity and microglial inflammatoryresponse at the injury site. Specifically, three weeks after SCI, the 5 injections of the combined cBGS (comb 5 injection) treatment induced a significant reduction in inflammatory response, as demonstrated by statistically significant lower levels of astrocyte activation (GFAP) compared to the control untreated mice.

[0473] Thus, it supported the herein disclosed conclusion that combined cBGS treatment mediated attenuation of inflammatory response at the lesion site and development of glial scarring also when injected even 8 hours after injury.

[0474] These results further indicate that combined cBGS treatment improves lesioned area condition and cell survival by reducing excitotoxicity in the eight hours therapeutic window.

[0475] In a further assay, myelin breakdown, synaptic degeneration, and neuronal death at the lesion site were examined, following treatment with the combined cBGS, with a first injection 8 hours post-injury, followed by 4 daily treatments, as compared to the non-treated group.

[0476] As shown in Figure 10A, three weeks after spinal cord compression, a significant reduction of myelin basic protein breakdown at the injury site of combined 5-day cBGS (comb 5inj) treated mice was observed. This result shows that cBGS treatment significantly reduces myelin breakdown at the injury site, followed by axonal survival, even when the first injection is administered 8-hour post injury (belated / late therapeutic window)

[0477] Motoneuron survival was demonstrated by analysis of NeuN’s large positive cell count at the lesion site up to 1mm. As shown in Figure 10B, a significantly higher number of NeuN-positive cells was observed in combined cBGS (comb 5inj) treated mice compared to vehicle control. Moreover, combined cBGS (comb 5inj) treatment significantly reduced synaptic degeneration around motor neurons at the lesion site, as shown in Figure 10C. These results align with the improved hindlimb motor function in the 5-day combined cBGS (comb 5inj) treatment group compared to the control.

[0478] The data presented clearly shows that a single injection of combined BGS treatment significantly improved motor functions over a single or 5-injections treatment with either of the enzymes (rGOTl / OxAc / PLP or rGPTl / Pyr / PLP).Moreover, the combined BGS treatment was effective even in the extended / belated therapeutic window of a first injection 8 hours after injury, which is very important for the clinical application of the treatment.

[0479] Example 7 - Traumatic Brain Injury

[0480] The effect of the combined BGS treatment was further investigated in another model of neurological damage, specifically traumatic brain injury (TBI). Specifically, the effect of the combined BGS treatment on the pro-inflammatory response and the motor and cognitive functions were examined in the TBI model.

[0481] Methods

[0482] Controlled cortical impact (CCI) model:

[0483] The open-head CCI model is frequently used to model TBI in rodents because of the ability to produce focal and precise cortical damage, low mortality rate, ability to create graded injury, the omission of skull fracture, intra-operator reproducibility, and low variability, resulting in the need for lower animal numbers.

[0484] Under isoflurane, 2%-0.5% anesthesia mice were attached to a stereotactic device of the impactor to hold the head stationary and align the impact device to the desired location. A midline incision was made to expose the skull, and a craniectomy was performed 2-2.5 mm posterior to bregma and 2-2.5 mm lateral to the midline suture, preserving dura mater integrity while minimizing cortical tissue perturbation.

[0485] Following the craniectomy, the impact device (2mm wide) was placed over the exposed dura mater with the appropriate coordinates. The injury was then induced with a specific cortical depression of 2mm depth, velocity 3m / s, and dwell time 0.5s. Then, the skin was glued with M3bond bio glue, and mice were placed in a warmed chamber for recovery, with daily assessments of health indicators and daily analgesic injections for 3 days. These defined locations result in damage to the primary motor cortex, the medial and lateral parietal association cortex, and are located directly above the septal pole of the hippocampus.

[0486] Treatment for moderate TBI experiments

[0487] The mice were treated with a cBGS combination having the formulation:cBGS'. rGOTl (59 mg / ml) + OxAc (0.03M) combined with rGPTl (21 mg / ml) + 0.03M Pyr + PLP (5 pM).

[0488] The cBGS combination was prepared by first mixing the substrates Pyr and OxAc, which mixture was adjusted to pH 5.5-6.0 using NaOH, and adding to the mixture the rGOTl and GPT enzymes.

[0489] Each treated mouse received one i.v. injection of a 200μl bolus of the cBGS treatment starting 1 hour post-TBI, followed by i.p injection for two or four days after the injury. Each bolus contained 30pg (59 mg / ml) of rGOTl with OxAc (0.03M) and 50 pg (21 mg / ml) of rGPTl with Pyr (0.03M) plus PLP (5 pM). As a control, mice were injected with 200μ μL iv 0.9% saline followed by a daily i.p injection.

[0490] Brain tissue was embedded in optimal cutting temperature compound (OCT) and cryostat-sectioned at thicknesses of 60 pm and 20 pm.

[0491] A quantitative analysis in different tissue thicknesses was carried out to examine different patterns of glial activation. Quantitative analysis was conducted on sections of different thicknesses to explore distinct patterns of glial activation. Sections at 20 pm enabled close examination of expression near the lesion site, while 60 pm sections minimized background signal and facilitated the detection of subtle differences in immunofluorescence. Staining methods varied: sections at 60 pm were stained using the free-floating technique, whereas sections at 20 pm were slide-mounted. Sections (at least 5 sections from each mouse) were imaged by fluorescence microscopy using an Axioplan Z1 (Zeiss) epifluorescence microscope. Photomicrographs (1300 x 1030 dpi) were obtained with 10x and 20x Plan-Neofluar (Zeiss, Germany) objectives, and acquired using an AxioCam (Zeiss, Germany) digital camera using AxioVision software (v. 4.4; Zeiss, Germany). Images were edited and colored using Adobe Photoshop 11, and density analyses were completed using ImageJ. Axons crossing the lesion site were counted manually in a double-blind way. NeuN was also visualized using the SP8 LIGHTNING confocal microscope (Leica Microsystems).

[0492] RT-qPCR of brain tissue

[0493] Quantitative reverse transcription PCR (RT-qPCR) was used to analyze the DNA quantification of IL-ip and CX3CR1 cytokines (qPCR Thermal Cycler, Quantabio). After transcardiac perfusion with PBS and brain dissection, tissue samples were immediatelyfrozen in liquid nitrogen and stored at -80°C. RNA extraction was performed using the Quick-RNA Mini-Prep kit (Zymogen) according to the manufacturer's protocol. The extracted mRNA was reverse transcribed into cDNA using the QScript Reverse Transcription System kit (QuantaBio) and a PCR machine. The samples were analyzed in triplicates using an RT-PCR system (QuantStudio Applied Biosystems) and quantified using the ΔΔCt method with GAPDH as the internal control gene. The following primers were used:

[0494] GAPDH

[0495] forward 5'- CCA GAA CAT CAT CCC TGC -3' (SEQ ID NO: 8)

[0496] reverse 5'- GGA AGG CCA TGC CAG TGAGC -3' (SEQ ID NO: 9)

[0497] IL-ip

[0498] forward 5'- ACC CCA AAA GAT GAA GGG CT -3' (SEQ ID NO: 10)

[0499] reverse 5'- GAT ACT GCC TGC CTG AAG CTCT -3' (SEQ ID NO: 11)

[0500] CX3CR1

[0501] forward 5'- CAG CAT CGA CCG GTA CCT T -3' (SEQ ID NO: 12)

[0502] reverse 5'- GCT GCA CTG TCC GGT TGT T-3' (SEQ ID NO: 13)

[0503] Motor &cognitive tests:

[0504] Grip test, beam walking test, and open field test were conducted as follows:

[0505] Beam walking test

[0506] The beam apparatus consists of two 1-meter wooden beams: one round with a diameter of 0.7 cm and one rectangular with a diameter of 1 cm. The apparatus is elevated 50 cm above the tabletop on two poles. At the end of each beam, a brown box serves as the finish point, and below the beams, a sponge pad is suspended in case of any falls. Prior to injury, mice underwent 3 training sessions (one per day), where they were placed at a starting point and guided across the beam until they could cross it reliably. Between training or testing trials, the beams and boxes were cleaned of mouse droppings using towels soaked in 70% ethanol and water before the next mouse was tested. During testing, mice were placed on beams, and the average crossing time over three trials was calculated for each beam at each testing time point. The timer startedwhen the mouse took its first step along the beam and stopped when the animal reached the safe box.

[0507] Grip strength test

[0508] The grip strength test was utilized to measure forelimb muscle strength as an indicator of neuromuscular function. The grip strength determines the maximal peak force displayed by an animal when pulling out the metal bar. The grip strength meter is positioned horizontally, and the subjects are held by the tail and lowered towards the apparatus. The Grip Strength Meter (Ugo Basile Cat. 47200) was set up horizontally on a stable table. During the test, each mouse was held by the tail and lowered towards the apparatus, allowing it to grasp the metal bar with its front paws. Once the optimal grip was established, the mouse was gently pulled backward by the tail in the horizontal plane at a constant speed until the grip was released. The peak tension was recorded as the mouse released its grip. The test consisted of three successive trials, with data collected and averaged for each group of mice.

[0509] Open field

[0510] Mice were acclimated to the testing room for at least 30 minutes before testing to minimize stress- related behavioral effects. After this acclimation period, each mouse was individually placed in the center of a 100 cm * 100 cm open field box to explore for 15 minutes. An overhead video camera recorded their movements. For each mouse, the time spent in the 50 cm x 50 cm center region of the box was recorded, as well as the total distance traveled within the box.

[0511] Results

[0512] In the subacute phase of Traumatic Brain Injury (TBI), activated glial cells are involved in the production of pro-inflammatory cytokines, which play critical roles in the secondary injury following primary brain injury. Therefore, it was tested whether the combined BGS treatment could affect the subacute inflammation in a TBI model.

[0513] As shown in Figures 11A-11B, the combined cBGS treatment reduced pro-inflammatory markers in the sub-acute phase after moderate TBI.

[0514] Specifically, Figures 11A-11B show that significantly lower levels of the pro-inflammatory cytokines, IL-ip (Figure 11 A) and CX3CR1 (Figure 11B) were detectedby qPCR in the combined BGS (cBGS) treated group compared to the control group. IL-ip is a pro-inflammatory cytokine produced by activated immune cells, driving inflammation. CX3CR1 is a chemokine receptor essential for the migration and activation of immune cells in inflammatory processes. Together, they play significant roles in modulating immune responses and inflammation.

[0515] These findings indicate that combined BGS treatment is effective in decreasing pro-inflammatory response and reducing secondary injury after TBI.

[0516] In addition, the combined BGS treatment improved the mice motor and cognitive functions as evident from Figures 12A-12C.

[0517] Specifically, the beam walking test is a popular test for analyzing mice's gait in an environment that challenges their ability to balance themselves. The grip strength test was utilized to measure forelimb muscle strength as an indicator of neuromuscular function. The grip strength determines the maximal peak force displayed by an animal when pulling out the metal bar. For cognitive-behavioral assessment, the open field test was applied.

[0518] The sensitive grip strength test was used to assess neuromuscular function by measuring the maximal muscle strength of the forelimbs 5 days post-injury. As shown in Figure 12A, the combined c7> GS-treated mice exhibited significantly higher grip strength compared to control mice, and their performance was equal to that of uninjured mice (“naive”), suggesting a high efficiency of the treatment on neuromuscular recovery and muscle function post-injury.

[0519] Further, using the beam walking test 24 hours post- TBI, fine motor coordination deficits were observed in injured mice compared to naive mice. As shown in Figure 12B, c7> GS-treated mice exhibited a 26% improvement in beam walking performance compared to the control group, suggesting that cBGS treatment holds promise for enhancing motor function following TBI.

[0520] In the open field test, the results of which are presented in Figure 12C, the combined BGS treatment group showed an increase in time spent in the center zone compared to controls, indicating a reduction in anxiety-like behavior in mice 5 days post-TBI.-n - Example 8 - Traumatic Brain Injury - neuronal survival

[0521] Based on the promising effects of the combined BGS treatment in reducing the pro-inflammatory response and mitigating potential secondary injury after TBI, along with improving motor and cognitive functions, its effect on neuronal survival was subsequently evaluated.

[0522] Methods

[0523] Mice: TgN (Thyl-EYFP) transgenic mice

[0524] Treatment protocol:

[0525] cBGS'. rGOTl (1 mg / kg) + OxAc (0.03M) + rGPTl (1.5 mg / kg) + Pyr (0.03M) + PLP (5 pM).

[0526] The mice were treated with the combined formulation cBGS, in 200 microliters of 0.9% Saline, with a first injection one hour after the injury, followed by 4 intraperitoneal daily injections at the same doses.

[0527] Thyl expression assay:

[0528] To evaluate neurodegeneration in the ipsilateral hemisphere, the following experiments were conducted using the TgN (Thyl-YFP) transgenic mice, and the Thyl expression in the region surrounding the lesion site was quantified according to the following procedure: Brain tissue was embedded in optimal cutting temperature compound (OCT) and cryostat-sectioned at thicknesses of 60 pm and 20 pm. The quantitation of the percentage of Thyl-YFP positive cells at the lesioned area out of the number of cells in the parallel contralateral side. Sections were imaged by fluorescence microscopy with an Olympus IX83 microscope and Cell Sens Dimension software. Quantitative analysis of Thyl densities was conducted using ImageJ software in a double-blind manner.

[0529] MBP immunodensity:

[0530] It was determined by quantitative analysis of myelin basic protein density (MB in the white matter tracks of axons at the lesion site and on the contralateral side. Cryostat longitudinal floating sections (60 pm) of fixed frozen brain tissue were stained using standard immunohistochemistry. A series of 60 pm-thick longitudinal sectionswere used for each antibody at the lesion site (10 slides from 6 animals from each group were analyzed). Sections were imaged by fluorescence microscopy with an Olympus IX83 microscope and CellSens Dimension software. Quantitative analysis of Thyl densities was conducted using Image J software in double blind way.

[0531] Inflammatory markers assay:

[0532] It was determined by immunohistochemistry as described above.

[0533] Results

[0534] It has been found that combined BGS treatment reduced neuronal degeneration at one week after moderate TBI. Specifically, as shown in Figure 13A and Figure 13B, compared to the control group (“CON”) the combined cBGS-treated group (“BGS”) exhibited a higher number of surviving neurons around the lesion site and reduced damage to the myeline as evident from the higher expression of MBP in survived Thy 1-YFP neurons.

[0535] Further, in the acute stage of TBI, cell damage, including myelin disruption, triggers an immune response by activating microglial cells, which respond to ATP release from injured neurons. Activated microglia subsequently release neurotoxic factors, with glutamate being a major contributor to neurodegeneration at the injury site. Microglial-derived glutamate induces excitotoxicity by activating N-methyl-D-aspartate (NMD A) receptors, leading to increased calcium influx and further neuronal damage. Astrocytes also react acutely to CNS injury by producing cytokines and chemokines. Studies have shown that elevated intracellular calcium levels in astrocytes can trigger glutamate release, exacerbating excitotoxicity and contributing to neuronal death. In the chronic stage, prolonged inflammatory responses create a pro-inflammatory brain microenvironment that exacerbates secondary cell death and accelerates neurodegeneration following TBI. Thus, the inflammatory response following TBI induces excitotoxicity, triggering a cascade of cellular and molecular events. This cascade drives neuronal damage and significantly amplifies the inflammatory response, leading to a vicious cycle of injury and inflammation. This ongoing cycle contributes to prolonged neurodegeneration and worsens the overall impact of TBI, highlighting the need for effective interventions targeting bothinflammatory and excitotoxic processes. Therefore, inflammatory markers were also analysed following treatment.

[0536] Specifically, as shown in Figures 14A-14B, the combined BGS treated group (“BGS”) demonstrated decreased astrogliosis, indicated by reduced GFAP density (Figure 15A) and decreased microglial density, indicated by reduced Ibal density (Figure 15B), around the lesion site compared to the control group. These results demonstrate again the strong neuroprotective effect of combined BGS treatment and its potential to enhance recovery post-TBI.

[0537] Example 11 - Effect of cBGS on repetitive mild traumatic brain injury (rmTBI)-Athletic Concussion

[0538] Another model used for testing the effect of the combined BGS treatment was the repetitive mild traumatic brain injury (rmTBI) - which is commonly used as a model to study athletic concussion. This model is particularly valuable for investigating the cumulative effects of repeated head impacts, which are characteristic of sports-related concussions.

[0539] Methods

[0540] Rats: Female Sprague Dawley rats (n = 24; 250-270 g) were obtained from Charles River (Worcester, MA). Adult male and female rats were subjected to 3 head impacts separated by 24 hrs. under anesthesia. Sham controls were subjected to three exposures of anesthesia separated by 24 hrs. to reflect the rmTBI protocol. The impact piston was directed to the top of the skull, midline, in the approximate area of Bregma. All vehicle and TBI rats were anesthetized with 2% isoflurane while being put in a restraint cone and head holder, then put into the impactor holder and let to wake up, then hit. Rats were awake and ambulatory within 1-2 min after the concussion. The impact was created repetitively over the course of three days, with 24 hours between the three given impacts. This regimen produced no signs of contusion. Rats were observed twice daily, in the morning and early evening, over the course of the regimen. There were no unplanned mortalities over the course of the study.Treatment groups

[0541] Combined cBGS Oxaloacetate (0.03M), pyruvate (0.03M), rGOTl (300mcg / rat) and rGPTl (500mcg / rat) + PLP (5 pM) in 1 ml of 0.9% Saline.

[0542] Control: sterile saline, isotonic solution, 0.9%)

[0543] Repetitive Mild Head Injury

[0544] Working with engineers at Animals Imaging Research, LLC (Holden, MA, United States), a pneumatic pressure drive was replicated, a 50 g compactor described by Viano D. C. et al., Neurosurgery, 2009, 64: 1162-1173, and reliably produced the 7.4, 9.3, and 11.2 m / s impact velocities described for mild, medium, and severe rat head injury, respectively. On the basis of this model, the behavioral effects of mild TBI controlling for the axis of injury, rotational force, and head acceleration in different directions were tested (Mychasiuk R. et al., J. Neurosci. Methods, 2016: 257 168–178). The impact created linear acceleration with some rotation. In the following experiment, 7.4 m / s impact velocities as determined using light-gate measured recordings were used. The impact piston was directed to the top of the skull, midline, in the approximate area of Bregma. All vehicle and TBI rats were anesthetized with 2% isoflurane while being put in a restraint cone and head holder, then put into the impactor holder and let to wake up, then hit. Rats were awake and ambulatory within 1-2 min after concussion. The impact was created repetitively over the course of three days, with 24 hours between the three given impacts. This regimen produced no signs of contusion. Rats were observed twice daily, in the morning and early evening, over the course of the regimen. There were no unplanned mortalities over the course of the study.

[0545] Drug and Head Impact Regime

[0546] Rats were acclimated to the animal colony 5 days prior to any experimental manipulations. Beginning on about postnatal day 70, rats were randomly divided into 2 groups. One group being Vehicle (n = 8) and one group being combined cBGS treatment (n=8) and began their head impact and drug regimens. Both groups received all three head impacts using a custom setup from Animal Imaging Research (AIR; Holden, MA). With a delay of 15 minutes, 0.5mL of the vehicle (sterile saline, isotonic solution, 0.9%) and 0.5mL the BGS treatment (Oxaloacetate (0.03M), pyruvate(0.03M), rGOTl (300mcg / rat) and rGPTl (500mcg / rat) + PLP (0.008pM)) were given intravenously post each hit.

[0547] Magnetic Resonance Imaging

[0548] Imaging System and anatomy acquisition

[0549] Imaging sessions were conducted on the day of the third impact an hour after the procedure, using a Bruker Biospec 7.0T720-cm USR horizontal magnet (Bruker, Billerica, MA, United States) and a 20-G / cm magnetic field gradient insert (ID = 12 cm) capable of a 120-ps rise time. Radio frequency signals were sent and received with a quadrature volume coil built into the animal restrainer (Animal Imaging Research, Holden, MA, United States). The design of the restraining system included a padded head support obviating the need for ear bars helping to reduce animal discomfort while minimizing motion artifact. All rats were imaged under 1-2% isoflurane while keeping a respiratory rate of 40-50 / min. At the beginning of each imaging session, a high-resolution anatomical data set was collected using the RARE pulse sequence with following parameters, 35 slice of 0.7 mm thickness; field of view (FOV) 3 cm; 256 x 256; repetition time (TR) 3900 ms; effective echo time (TE) 48 ms; NEX 3; 6 min 14 s acquisition time.

[0550] Diffusion Weighted Imaging - Quantitative Anisotropy

[0551] Diffusion weighted imaging was acquired with a spin-echo echo-planar-imaging (EPI) pulse sequence having the following parameters: TR / TE = 500 / 20 ms, eight EPI segments, and 10 non-collinear gradient directions with a single B-value shell at 1000 s / mm2 and one image with a B-value of 0 s / mm2 (referred to as B0). Geometrical parameters were: 48 coronal slices, each 0.313 mm thick (brain volume) and with in-plane resolution of 0.313 x 0.313 mm2 (matrix size 96 x 96; FOV 30 mm2). The imaging protocol was repeated two times for signal averaging. Each DWI acquisition took 35 min and the entire MRI protocol lasted ca. 70 min. Image analysis included DWI analysis of the DW-3D-EPI images to produce the maps of fractional anisotropy (FA) using a 3D MRI Rat Brain Atlas©(Ekam Solutions LLC, Boston, MA, United States). DWI analysis was completed with MATLAB and MedINRIA (1.9.01) software. Because sporadic excessive breathing during DWI acquisition can lead to significant image motion artifacts that are apparent only in the slices sampled whenmotion occurred, each image (for each slice and each gradient direction) was screened, prior to DWI analysis. If found, acquisition points with motion artifacts were eliminated from analyses.

[0552] For statistical comparisons between rats, each brain volume was registered to the 3D rat atlas allowing voxel- and region-based statistics. All image transformations and statistical analyses were carried out using the in-house MIVA software2. For each rat, the BO image was co-registered with the BO template (using a six-parameter rigid-body transformation). The co-regi strati on parameters were then applied on the DWI indexed maps for the different indices of anisotropy. Normalization was performed on the maps since they provided the most detailed visualization of brain structures and allowed for more accurate normalization. The normalization parameters were then applied to all DWI indexed maps that were then smoothed with a 0.3-mm Gaussian kernel. To ensure that FA and RD values were not affected significantly by the preprocessing steps, the “nearest neighbor” option was used following registration and normalization.

[0553] Statistical differences in measures of DWI between experimental groups were determined using a nonparametric Mann-Whitney U-test (alpha set at 5%), according to Kulkami P. et al., [Front SystNeurosci. 2019; 13:34],

[0554] Results

[0555] The results presented hereinbelow show that cBGS treatment decreased white matter damage after repeated mild traumatic brain injury (r-m TBI). As described above, adult male and female rats were subjected to 3 head impacts separated by 24 hrs. under anesthesia. Sham controls were subjected to three exposures of anesthesia separated by 24 hrs. to reflect the r-mTBI protocol.

[0556] Neuropathology of the brain was followed with Diffusion Weighted Imaging-Quantitative Anisotropy immediately after the third brain impact. As shown in Figures 15A-15C, areas of the basal ganglia (Figure 15A), cortex (Figure 15B), and prefrontal cortex (Figure 15C) show significant differences in fractional Anisotropy (FA) values between vehicle and combined BGS-treated groups when compared to a non-treated control rat. These areas demonstrate higher FA values, which is indicative of a larger white matter presence and suggest a white matter decay in the vehicle-treatedgroup post-r-mTBI, but not when treated with combined BGS. Moreover, the combined BGS treatment group demonstrated similar levels of FA values as vehicle naive rats, demonstrating the ability of the treatment to completely prevent the damage to white matter induced by rm TBI.

[0557] Further, the combined BGS treatment decreased neuroinflammation after r-m TBI as evident from Figures 16A-16D.

[0558] The combined BGS treatment was administered 15 minutes after each brain impact, and microglia activation was assessed by measuring the activated Ibal density, a marker for microglia / macrophage.

[0559] As shown in Figures 16A-16D, a reduction in microglia density was observed at the Prefrontal cortex (Figures 16A-16B) and Hippocampus (Figure 16C-16D) of the combined BGS treatment group. Thus, it was concluded that BGS treatment mediated attenuation of inflammatory response after rm TBI.

[0560] Similarly, astrocyte reactivity was assessed by measuring the expression levels of GFAP, a marker for astrocytic reactivity in the various brain regions.

[0561] As shown in Figures 17A-17B, a reduction in astrocytic reactivity was observed at the hippocampus (Figure 17A) and hypothalamus (Figure 17B), of the combined BGS treatment group.

[0562] The results show that combined BGS treatment decreases inflammatory response after rm TBI. It is expected that the combined BGS will also be better than the effect of treatment with a single enzyme, with its substrate.

[0563] Example 12 - Effect of Single and Combined BGS Treatment at 4 Hours in a Traumatic Brain Injury Model

[0564] The therapeutic effect of single and combined BGS treatments was further investigated in the traumatic brain injury (TBI) model. In particular, the effects of BGS administration compared to the single enzyme and its substrate administered at 4 hours post-injury were evaluated. The studies examined the impact of BGS treatment on neurological and behavioral outcomes, including motor coordination and anxiety-like behavior, as well as on tissue-level pathological responses, such as edema formation,lesion size, neuronal survival, neuroinflammatory and glial responses, and white-matter integrity.

[0565] Methods

[0566] Controlled. cortical impact (CCI) model:

[0567] Male and female Thyl-YFP transgenic mice or their wild-type littermates were anesthetized with Isoflurane. The head was immobilized on a stereotaxic frame, and the skull was exposed by a midline incision. A craniotomy of 2-mm diameter was made using a microdrill and trephine over the right fronto-parietal cortex (relative to bregma, 1.0 mm anterior, 1.0 mm lateral), and the bone flap was removed without damage to the dura. Mice were then subjected to CCI using a pneumatic impactor apparatus with a 2- mm round-tip impactor. Zero position was set when the tip touched the dural surface under the guidance of a surgical microscope. The velocity of impact was set at speed of 2 m / s, with an impact depth of 2 mm and a dwell time of 2 sec. Immediately after the injur} / , the skin was closed with VetBond glue.

[0568] Treatment for TBI experiments

[0569] The following treatment groups were included:

[0570] cBGS formulation: rGOTl (30pg) + OxAc (0.03M) + rGPTl (50pg) + Pyr (0.03M) + PLP (0.008pM) in 200 pl per one mouse

[0571] rGOTl formulation: rGOTl (30pg) + OxAc (0.03M) + PLP (0.008pM) in 200 pl per one mouse.

[0572] rGPTl formulation: rGPTl (50pg) + OxAc (0.03M) + PLP (0.008pM) in 200 pl per one mouse.

[0573] Animals received a total of five intravenous injections, consisting of an initial injection administered 4 hours post-injury, followed by four once-daily subcutaneous injections of the same formulation.

[0574] Control group: Animals received one intravenous and four subcutaneous injections of 0.9% saline, including an initial injection at 4 hours post-injury, followed by four once-daily injections.Sham group: mice anesthetized, and the skin was cut without inducing TBI and injected with vehicle (0.9% Saline).

[0575] MRI

[0576] Magnetic Resonance Imaging (MRI) was used to assess the edema and lesion size of the injured mice at various time points (24 hours, 48 hours, and 7 days) post¬ injury. Mice were anesthetized with ~ 2% isoflurane in oxygen throughout the MRI experiments. Body temperature was monitored and maintained at 38 °C with a warm water circulation system. MRI experiments were performed on a 7T / 30 MRI Biospec Broker scanner (Broker, Germany) equipped with a gradient system of 600 m’T / m at the Strauss Computational Neuroimaging Center in Tel Aviv University. For excitation, a body-coil (outer / inner diameter of 112 / 86 mm) was used and a quadrate coil (10 mm diameter) served as a receiver.

[0577] For anatomical images, T2-weighted contrast was acquired using a rapid acquisition with refocusing echoes (RARE) sequence with the following parameters: TR / TE = 3000 / 30ms, 4 averages, and RARE factor = 10. 28 coronal slices of 500 microns ensuring full brain coverage (no gaps), with in-plane resolution of 100x100 um2. Imaging lasted about 3min,

[0578] Images were analyzed using in-house MATLAB (MathWorks) code to manually mark the edema and lesion sizes, and Weasis v4.6.5 was used to process the images.

[0579] Beam walking test and anxiety-like behavioral

[0580] Motor coordination was assessed using the Beam Balance Score (BBS). Mice were trained to traverse a 1-cni wide, 70-cm long square beam, and performance was recorded across three consecutive trials, with the mean value used for analysis. The BBS evaluated the animal’s motor function while traversing the beam- percent of hindlimb missteps was quantified as an index of motor impairment. Missteps were defined as any instance where a paw slipped off the beam, and the percentage was calculated as: (number of missteps / total steps) x 100.

[0581] Exploratory and anxiety-like behavior was assessed using the Circle Exit Test also known as the Anxiety Test. Each mouse was placed in the center of a circular arena, and the latency to exit the circle was recorded as a measure of spontaneous exploration and avoidance behavior. The behavioral assessments, including BBS performance,percent missteps, and Circle Exit Test latency, were conducted at the designated post¬ injury time points of 1 day (24 h), 2 days (48 h), 7 days, and 14 days after surgery.

[0582] Immunohistochemistry

[0583] Mice were perfused with PBS followed by 4% paraformaldehyde (PF A). Brains were removed and post-fixed for 1 hour in cold 4% PF A, followed by 20% sucrose in PBS overnight at 4 °C. Cryostat coronal floating sections (60 pm) of fixed frozen tissue were stained using standard immunohistochemistry. Primary antibodies: rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, Dako); rabbit anti-Ibal (1:500; Abeam); and mouse anti-CS56 (1:2500, Abeam). Secondary antibodies: Alexa Fluor 488 or 568; 1:1000 (Invitrogen). Sections were visualized by an Olympus IX83 fluorescence microscope with CellSens Dimension software. Fluorescence densitometry was measured in Images around the lesion site using by ImageJ blindly.

[0584] Statistics

[0585] Statistical analysis was performed using one-way ANOVA with subsequent Tukey’s test or a two-tailed West using Prizm software. Data is reported as mean ± STDEV. Differences were considered significant when P < 0.05.

[0586] Results

[0587] cBGS treatment prevented brain edema and significantly reduced injury size in the TBI mouse model. To assess the effect of different treatment groups administered 4 hours post- TBI on brain injury outcomes, magnetic resonance imaging (MRI) was used to evaluate brain edema and lesion size.

[0588] T2-weighted MRI analysis revealed marked differences in injury severity between cBGS-treated mice and saline-treated controls. At 24 hours post-injury (Figure 18A, top row) and 48 hours post-injury (Figure 18A, middle row), saline-treated mice exhibited extensive cerebral edema, whereas cBGS-treated (combined-treatment) mice showed substantially smaller edema areas. By 1 week post-injury (Figure 18A, bottom row), control mice demonstrated further lesion expansion and persistent edema, while cBGS-treated mice exhibited markedly attenuated edema and limited lesion progression.

[0589] Consistent with the representative MRI images, quantitative analyses demonstrated that the combined BGS treatment significantly reduced edema volume at24- and 48-hours post-injury (Figure 18B-18C) and lesion size at 1 week post-injury (Figure 18D) compared with saline-treated controls. Notably, cBGS treatment also resulted in significantly greater reductions in edema and lesion size compared with administration of each individual enzyme in combination with its respective cosubstrate and PLP.

[0590] Together, these results indicate that combined BGS treatment more effectively mitigates early tissue damage and attenuates the progression of secondary injury over time than treatment with individual enzyme-based formulations.

[0591] cBGS treatment improves neurological, motor, and anxiety-related behavioral outcomes following traumatic brain injury

[0592] To assess the effect of cBGS treatment administered 4 hours post-injury on functional recovery following traumatic brain injury, behavioral performance was evaluated across multiple assays. At 48 hours post-injury, cBGS-treated mice exhibited a marked reduction in the percentage of missteps on the 1-cm beam-walking test compared with saline-treated controls and mice receiving individual enzyme-based treatments (Figure 19A), indicating improved early motor coordination and reduced neurological impairment.

[0593] At 1 week post-injury, anxiety-related exploratory behavior was assessed using the anxiety circle test. Saline-treated TBI mice displayed restricted exploratory activity, consistent with increased anxiety-like behavior. In contrast, cBGS-treated mice exhibited normalized exploratory behavior comparable to non-injured controls, indicating attenuation of anxiety-like symptoms (Figure 19B).

[0594] Together, these findings demonstrate that cBGS treatment initiated 4 hours postinjury more effectively improves early neurological and motor function and reduces anxiety-like behavior during the subacute recovery phase following traumatic brain injury compared with other treatment groups.

[0595] cBGS treatment promotes neuroprotection following traumatic brain injury and attenuates glial responses

[0596] To assess neuronal survival, the density of Thyl-YFP-positive neurons, expressed in cortical motor neurons within the injured region, was quantified. Mice treated for five consecutive days with cBGS exhibited significantly higher Thyl-YFPdensity in the lesioned area compared with saline-treated controls, and mice treated with rGOTl + OxAc + PLP, or rGPTl + Pyr + PLP treated groups (Figure 20A). Among all treatment groups, cBGS treatment resulted in the greatest preservation of neuronal cell bodies within the lesion core. Importantly, these outcomes were consistent across animals and aligned with the functional improvements observed in behavioral assays, supporting the robust neuroprotective capacity of cBGS following TBI.

[0597] Furthermore, mice treated with cBGS showed a significant increase in myelin basic protein (MBP) density at the injury site relative to the corresponding contralateral region (Figure 20B). Notably, only the combined cBGS treatment produced a statistically significant reduction in myelin loss, whereas treatment with each individual enzyme and its respective co-substrate failed to demonstrate a protective effect. Because myelin damage can lead to the release of myelin debris that promotes microglial activation, these findings indicate that cBGS treatment improves the local tissue environment at the lesion site and effectively inhibits axonal damage following traumatic brain injury.

[0598] Cell death, excitotoxicity, and inflammatory processes following neurotrauma are key drivers of astrocyte reactivity and glial scar formation. To assess whether cBGS or other treatment combinations modulate these responses, astrocyte activation was evaluated by measuring GFAP expression in the perilesional region of mice treated with different combinations, with the first injection administered 4 hours after moderate to severe TBI.

[0599] Quantitative immunohistochemical analysis performed one month post-injury demonstrated that animals treated for five consecutive days with cBGS, exhibited significantly reduced astrogliosis compared with saline-treated controls, mice treated with rGOTl + OxAc + PLP, or rGPTl + Pyr + PLP, as evidenced by decreased GFAP density at the lesion borders (Figure 21A). Notably, cBGS treatment produced the most pronounced reduction in GFAP expression, exceeding the effects observed with either individual enzyme-based treatment, indicating a stronger attenuation of astrocyte reactivity.

[0600] To further evaluate the inflammatory response, microglial activation was assessed by quantifying Ibal expression, a marker of activated microglia andmacrophages. One month after TBI, Ibal immunostaining revealed a significant reduction in microglial density in the perilesional area only in mice treated with cBGS, compared with saline controls and mice treated with rGOTl + Ox Ac + PLP or rGPTl + Pyr + PLP (Figure 21B). These results indicate that cBGS treatment initiated 4 hours post-injury exerts the strongest anti-inflammatory effect among the tested treatment groups.

[0601] Finally, the expression of chondroitin sulfate proteoglycans (CSPGs) was evaluated using CS56 immunostaining. CSPGs are secreted by astrocytes in the perilesional region and form a chemical barrier that inhibits axonal regeneration and induces growth cone collapse. Consistent with the reduction in astrocyte activation, a significant decrease in CSPG expression was observed, with the most pronounced inhibition detected in the cBGS-treated group (Figure 21C), indicating a more permissive environment for axonal regeneration.

[0602] Example 13: Effect of Single and Combined BGS Treatment at 8 Hours in a Traumatic Brain Injury Model

[0603] To determine whether delayed treatment initiation preserves functional benefits, neurological and motor outcomes were evaluated in mice receiving different treatment combinations, with the first injection administered 8 hours post-injury. Motor coordination was assessed using the percentage of missteps on a 1-cm beam-walking test at 24- and 48-hours post-injury.

[0604] As shown in Figure 22A-22B, only mice treated with cBGS exhibited a significant reduction in missteps compared with saline-treated controls and mice receiving individual enzyme-based treatments, indicating improved motor precision and reduced neurological deficits. Notably, the magnitude of motor improvement observed with 8-hour cBGS treatment was comparable to that achieved with treatment initiated at 4 hours post-injury (Figure 19A), demonstrating preservation of therapeutic efficacy despite delayed administration.

[0605] Collectively, these findings demonstrate that cBGS treatment remains effective when initiated up to 8 hours after TBI, resulting in significant improvement in early neurological status and motor coordination. Moreover, these results support the efficacyof cBGS when administered at a clinically relevant delayed time point, underscoring its potential to promote functional motor recovery following TBI.

[0606] To further assess neuroprotective effects, neuronal survival was quantified by measuring the percentage of Thyl-YFP-positive cells in the lesioned region compared with the contralateral area. Mice treated for five consecutive days with cBGS exhibited a significantly higher level of neuronal survival at the injury site compared with saline-treated controls and mice treated with rGPTl + Pyr + PLP (Figure 23A). Treatment with rGOTl + OxAc + PLP showed a trend toward reduced neuronal loss relative to controls; however, this effect was less pronounced than that observed with cBGS. In addition, mice treated with cBGS, with treatment initiated 8 hours post-injury, exhibited a significant preservation of myelin basic protein (MBP) immunoreactivity, whereas treatment with individual enzyme-co-substrate formulations failed to prevent myelin loss (Figure 23B), indicating that only cBGS effectively limits axonal and myelin damage following TBI.

[0607] Overall, cBGS treatment resulted in the greatest preservation of neuronal cell bodies within the lesion core and attenuated axonal and myelin damage, supporting its robust neuroprotective capacity when treatment is initiated 8 hours after injury.

[0608] To determine whether cBGS or individual enzyme-based treatments reduce astrocyte activation and glial scarring when administered at a delayed time point, GFAP expression was examined in the perilesional region of mice subjected to moderate-to-severe TBI, with the first injection administered 8 hours post-injury. Animals treated for five consecutive days with cBGS produced the greatest attenuation of GFAP expression, demonstrating the most robust suppression of astrocyte activation and glial scarring (Figure 24A).

[0609] Furthermore, quantitative analysis of Ibal immunoreactivity demonstrated that microglial density in the perilesional area was significantly reduced only in mice treated with cBGS for five consecutive days (Figure 24B), compared with vehicle-treated controls. Neither of the individual enzyme-based treatments produced comparable reductions in microglial accumulation.

[0610] Collectively, these findings demonstrate that when treatment is initiated at a clinically relevant delayed time point of 8 hours, only the combined cBGS therapyretains robust therapeutic efficacy, significantly reducing astrocyte activation, preserving neuronal survival, and attenuating post-injury neuroinflammation in a TBI mouse model.

Claims

CLAIMS:

1. A combination for use in treatment of a subject in need thereof, the combination comprising the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the first transaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, the components being formulated for administration either as a single combined composition or as separate compositions.

2. The combination for use of claim 1, wherein the first transaminase and the second transaminase are selected from the group consisting of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

3. The combination for use of claim 1 or 2, wherein the first substrate to the first transaminase and the second substrate to the second transaminase are selected from the group consisting of pyruvate (Pyr) and oxaloacetate (OxAc).

4. The combination for use of any one of claims 1 to 3, wherein the co-factor is pyridoxal phosphate (PLP).

5. The combination for use of any one of claims 1 to 4, comprising administration of a combination comprising GPT as the first transaminase, GOT as the second transaminase, Pyr as the first substrate to the first transaminase, OxAc as the second substrate to the second transaminase, and PLP as the co-factor.

6. The combination for use of any one of claims 1 to 5, wherein the treatment is of neurological damage caused by trauma or injury.

7. The combination for use of any one of claims 1 to 6, comprising at least a first administration of the combination and optionally at least one additional administration.

8. The combination for use of claim 7, wherein the first administration of the combination is at a first time point after the trauma, the first time point being within an early treatment window comprising any time point of up to 4 hours post trauma or injury.

9. The combination for use of claim 7, wherein the first administration of the combination is at the first time point and the at least one additional administration ofthe combination after the first treatment window, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration.

10. The combination for use of claim 7, wherein the first administration of the combination at a first time point after the trauma or injury, the first time point within a late treatment window that is more than 4 hours post injury or trauma.

11. The combination for use of claim 9, wherein the late treatment window is up to 36 hours post injury or trauma.

12. The combination for use of claim 9 or 10, wherein the first administration is 6-12 hours post injury or trauma.

13. The combination for use of any one of claims 7 to 12, wherein the at least one additional administration of the combination is subsequent to the first administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration, and at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma.

14. The combination for use of claim 13, comprises two or more of the at least one additional administration, and the two or more additional administrations are daily administrations of said combination.

15. The combination for use of any one of claims 1 to 14, wherein the treatment is of a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, injury resulting from repetitive head impacts, including sub-concussive impacts, blast related neurological injury, haemorrhagic brain injury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition.

16. The combination for use of any one of claims 1 to 15, wherein the treatment is of a neurological condition that is involved with elevated glutamate levels.

17. A method of treating a subject in need thereof, the method comprising administering to the subject a combination comprising the following components (i) a first transaminase, (ii) a second transaminase, (iii) a first substrate to the firsttransaminase, (iv) a second substrate to the second transaminase, and (v) a transaminase co-factor, said combination being formulated for administration either as a single combined composition or as separate compositions.

18. The method of claim 17, wherein the first and second transaminase are different and selected from the group consisting of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT).

19. The method of claim 17 or 18, wherein the first substrate and second substrate are selected from the group consisting of pyruvate (Pyr) and oxaloacetate (OxAc).

20. The method of any one of claims 17 to 19, wherein said co-factor is pyridoxal phosphate (PLP).

21. The method of any one of claims 17 to 20, comprising administration of a combination comprising GPT as said first transaminase, GOT as said second transaminase, Pyr as said first substrate to said first transaminase, OxAc as said second substrate to said second transaminase, and PLP as said co-factor.

22. The method of any one of claims 17 to 21, for treatment of neurological damage caused by trauma or injury.

23. The method of any one of claims 17 to 22, comprising at least a first administration of the combination and optionally at least one additional administration.

24. The method of claim 23, wherein the first administration of the combination at a first time point after the trauma or injury, the first time point being within a first treatment window of up to 4 hours post trauma or injury.

25. The method of claim 23, wherein the first administration of the combination is at a time point that is within a late treatment window, the late treatment window being greater than 4 hours post injury or trauma.

26. The method of claim 25, wherein the late treatment window is up to 36 hours post injury or trauma.

27. The method of claim 25 or 26, wherein the first administration is 6-12 hours post injury or trauma.

28. The method of any one of claims 23 to 26, wherein the at least one additional administration of the combination is subsequent to the first administration, the combination of the at least one additional administration being formulated the same or different from the combination of the first administration.

29. The method of any one of claims 17 to 28, comprising two or more of the at least one additional administration of the combination at respectively two or more additional time points after the first administration, and at least one of the two or more additional administrations is within a second treatment window of up to 48 hours post trauma.

30. The method of claim 29, wherein the two or more of the at least one additional administration are daily administrations of said combination.

31. The method of any one of claims 17 to 30, wherein the treatment is of a neurological damage selected from the group consisting of spinal cord injury (SCI), traumatic brain injury (TBI), concussion, blast brain injury, haemorrhagic brain injury, paraoxon intoxication, brain or cerebral hypoxia, stroke, ischemic stroke, cerebral palsy and an acute episode or seizure related to a neurological condition.

32. The method of any one of claims 17 to 31, for treatment of a neurological condition involved with elevated glutamate levels.

33. A pharmaceutical product comprising a combination of components:(i) a first transaminase(ii) a second transaminase,(iii) a first substrate to said first transaminase(iv) a second substrate to said second transaminase, and(v) a transaminase co-factor,wherein said components are formulated for administration either as a single combined pharmaceutical composition or as separate pharmaceutical compositions.

34. The pharmaceutical product of claim 33, wherein the first transaminase is GPT, the second transaminase is GOT, the first substrate to said first transaminase is Pyr, thesecond substrate to said second transaminase is OxAc and the transaminase co-factor is PLP.

35. The pharmaceutical product of claim 33 or 34, wherein, when the components are formulated for administration as separate pharmaceutical compositions, the first transaminase is in a same pharmaceutical composition with the first substrate and the second transaminase is in a same pharmaceutical composition with the second substrate.

36. The pharmaceutical product of any one of claims 33 to 35, comprising instructions for use of the combination of components in treating a neurological condition.

37. The pharmaceutical product of any one of claims 33 to 36, comprising instructions for use in treating neurological condition involved with elevated glutamate levels.

38. The pharmaceutical product of any one of claims 33 to 37, comprising instructions for use in treating neurological condition caused by trauma or injury.

39. The pharmaceutical product of any one of claims 33 to 38, wherein the instructions comprise steps for performing the method of any one of claims 17 to 32.

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